Pharmaceutical composition of nanoparticles

ABSTRACT

The invention discloses a pharmaceutical composition of bioactive nanoparticles composed of chitosan, poly-glutamic acid, and a bioactive agent for oral delivery. The chitosan-based nanoparticles are characterized with a positive surface charge and enhanced permeability for oral drug delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/799,283, filed Apr. 21, 2010, now U.S. Pat. No.7,910,086, which a continuation-in-part application of U.S. patentapplication Ser. No. 12/221,897, filed Aug. 7, 2008, now U.S. Pat. No.7,611,690, which is a continuation-in-part application of U.S. patentapplication Ser. No. 12/151,230, filed May 5, 2008, now U.S. Pat. No.7,541,046, which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/398,145, filed Apr. 5, 2006, now U.S. Pat. No.7,381,716, which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/284,734, filed Nov. 21, 2005, now U.S. Pat. No.7,282,194, which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/029,082, filed Jan. 4, 2005, now U.S. Pat. No.7,265,090, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is related to general uses of nanoparticles thathave a composition of chitosan and negatively charged substrate with atleast one bioactive agent and their enhanced permeability for oral orparenteral drug delivery.

BACKGROUND OF THE INVENTION

Production of pharmaceutically bioactive peptides and proteins in largequantities has become feasible (Biomacromolecules 2004; 5:1917-1925).The oral route is considered the most convenient way of administeringdrugs for patients or an animal subject. Nevertheless, the intestinalepithelium is a major barrier to the absorption of hydrophilic drugssuch as peptides and proteins (J. Control. Release 1996; 39:131-138).This is because hydrophilic drugs cannot easily diffuse across the cellsthrough the lipid-bilayer cell membranes. Attentions have been given toimproving paracellular transport of hydrophilic drugs (J. Control.Release 1998; 51:35-46). However, the transport of hydrophilic moleculesvia the paracellular pathway is, however, severely restricted by thepresence of tight junctions that are located at the luminal aspect ofadjacent epithelial cells (Annu. Rev. Nutr. 1995; 15:35-55). These tightjunctions form a barrier that limits the paracellular diffusion ofhydrophilic molecules. The structure and function of tight junctions isdescribed, inter alia, in Ann. Rev. Physiol. 1998; 60:121-160 and inBallard T S et al., Annu. Rev. Nutr. 1995; 15:35-55. Tight junctions donot form a rigid barrier but play an important role in the diffusionthrough the intestinal epithelium from lumen to bloodstream and viceversa.

Movement of solutes between cells, through the tight junctions that bindcells together into a layer such as the epithelial cells of thegastrointestinal tract, is termed paracellular transport. Paracellulartransport is passive. Paracellular transport is dependent onelectrochemical gradients generated by transcellular transport andsolvent drag through tight junctions. Tight junctions form anintercellular barrier that separates the apical and basolateral fluidcompartments of a cell layer. Movement of a solute through a tightjunction from apical to basolateral compartments depends on thepermeability of the tight junction for that solute.

Polymeric nanoparticles have been widely investigated as carriers fordrug delivery (Biomaterials 2002; 23:3193-3201). Much attention has beengiven to the nanoparticles made of synthetic biodegradable polymers suchas poly-ε-caprolactone and polylactide due to their biocompatibility (J.Drug Delivery 2000; 7:215-232; Eur. J. Pharm. Biopharm. 1995; 41:19-25).However, these nanoparticles are not ideal carriers for hydrophilicdrugs because of their hydrophobic property. Some aspects of theinvention relate to a novel nanoparticle system, composed of hydrophilicchitosan and poly(glutamic acid) hydrogels; the nanoparticles areprepared by a simple ionic-gelation method. This technique is promisingas the nanoparticles are prepared under mild conditions without usingharmful solvents. It is known that organic solvents may causedegradation of peptide or protein drugs that are unstable and sensitiveto their environments (J. Control. Release 2001; 73:279-291).

Following the oral drug delivery route, protein drugs are readilydegraded by the low pH of gastric medium in the stomach. The absorptionof protein drugs following oral administration is challenging due totheir high molecular weight, hydrophilicity, and susceptibility toenzymatic inactivation. Protein drugs at the intestinal epitheliumcannot partition into the hydrophobic membrane, leaving only theepithelial barrier via the paracellular pathway. However, the tightjunction forms a barrier that limits the paracellular diffusion ofhydrophilic molecules.

Chitosan (CS), a cationic polysaccharide, is generally derived fromchitin by alkaline deacetylation (J. Control. Release 2004; 96:285-300).It was reported from literature that CS is non-toxic and soft-tissuecompatible (Biomacromolecules 2004; 5:1917-1925; Biomacromolecules 2004;5:828-833). Additionally, it is known that CS has a special property ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells (Pharm. Res. 1994; 11:1358-1361).Most commercially available CSs have a quite large molecular weight (MW)and need to be dissolved in an acetic acid solution at a pH value ofapproximately 4.0 or lower, which is somewhat impractical. However,there are potential applications of CS in which a low MW would beessential. Given a low MW, the polycationic characteristic of CS can beused together with a good solubility at a pH value close tophysiological ranges (Eur. J. Pharm. Biopharm. 2004; 57:101-105).Loading of peptide or protein drugs at physiological pH ranges wouldpreserve their bioactivity. On this basis, a low-MW CS, obtained bydepolymerizing a commercially available CS using cellulase, is disclosedherein to prepare nanoparticles of the present invention.

Thanou et al. reported chitosan and its derivatives as intestinalabsorption enhancers (Adv Drug Deliv Rev 2001; 50:S91-S101). Chitosan,when protonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia.Co-administration of chitosan or trimethyl chitosan chloride withpeptide drugs were found to substantially increase the bioavailabilityof the peptide in animals compared with administrations without thechitosan component.

The γ-PGA, an anionic peptide, is a natural compound produced ascapsular substance or as slime by members of the genus Bacillus (Crit.Rev. Biotechnol. 2001; 21:219-232). γ-PGA is unique in that it iscomposed of naturally occurring L-glutamic acid linked together throughamide bonds. It is reported from literature that this naturallyoccurring γ-PGA is a water-soluble, biodegradable, and non-toxicpolymer. A polyamino carboxylic acid (complexone), such as diethylenetriamine pentaacetic acid, has showed enzyme resistant property. It isclinical beneficial to incorporate a PGA-complexone conjugate as anegative substrate and chitosan as a positive substrate in a drugdelivery nanoparticle formulation for better absorption performance withreduced enzymatic effect.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a novel, uniquenanoparticle system and methods of preparation for protein/peptide drugor bioactive agent delivery using a simple and mild ionic-gelationmethod upon addition of a poly-γ-glutamic acid (γ-PGA) solution (orother negatively charged component, such as PGA-complexion conjugate)into regular molecular weight chitosan (CS) solution. In one embodiment,the chitosan employed are N-trimethyl chitosan (TMC), low MW-chitosan,EDTA-chitosan, mono-N-carboxymethyl chitosan (MCC), chitosanderivatives, and combinations thereof that is positively charged. In oneembodiment, the molecular weight of a low-MW CS of the present inventionis about 80 kDa or less, preferably at about 40-50 kDa, adapted foradequate solubility at a pH that maintains the bioactivity of proteinand peptide drugs. It is stipulated that a chitosan particle with about30-50 kDa molecular weight is kidney inert. The particle size and thezeta potential value of the prepared nanoparticles are controlled bytheir constituted compositions. The results obtained by the TEM(transmission electron microscopy) and AFM (atomic force microscopy)examinations showed that the morphology of the prepared nanoparticles isgenerally spherical or spheroidal in shape. In one embodiment, thebioactive agent is a pegylated bioactive agent.

Some aspects of the invention relate to a method of enhancing epithelialpermeation (for example, intestinal or blood brain paracellulartransport) configured for delivering at least one bioactive agent,comprising administering nanoparticles composed of γ-PGA and chitosan,in an animal subject. Administering the nanoparticles may be via oraladministration, intranasal absorption, subcutaneous injection orinjection into a blood vessel. In one embodiment, the chitosan dominateson the surface of the nanoparticles as shell substrate and thenegatively charged γ-PGA or other suitable component, with chitosanpresent, as core substrate. In another embodiment, a substantial surfaceof the nanoparticles is characterized with a positive charge. In afurther embodiment, the nanoparticles of the present invention compriseat least one positively charged shell substrate and at least onenegatively charged core substrate. In one embodiment, all of thenegatively charged core substrate conjugates with a portion of thepositively charged shell substrate in the core portion so to maintain asubstantially zero-charged (neutral) core. In one embodiment, at leastone bioactive agent or protein drug is conjugated with the negativelycharged core substrate or the substantially zero-charged (neutral) core.

In a further embodiment, the nanoparticles have a mean particle sizebetween about 50 and 400 nanometers, preferably between about 100 and300 nanometers, and most preferably between about 100 and 200nanometers.

In some embodiments, the nanoparticles are loaded with a therapeuticallyeffective amount of at least one bioactive agent, wherein the bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, oxygen-enriching agent, oxygen-containing agent,anti-epileptic drug, and anti-inflammatory drugs. The anti-epilepticdrug may include Neurontin (gabapentin, a gamma-aminobutyric acidanalog), Lamictal (lamotrigine, shown to act at voltage-sensitive sodiumchannels, stabilizing neural membranes and inhibiting the release ofexcitatory neural transmitters), Febatol (felbamate, shown to have weakinhibitory effects on GABA receptor binding sites), Topamax (topiramate,has a novel chemical structure derived from D-fructose that blocksvoltage-sensitive sodium channels, enhances the activity of GABA, aninhibitory neurotransmitter, and blocks the action of glutamate, anexcitatory neurotransmitter), and/or Cerebyx (fosphenytoin, a phenyloinprecursor that is rapidly converted after parenteral administration).

Further, the bioactive agent may be selected from the group consistingof calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin,tyrotropin releasing hormone, follicle stimulating hormone, luteinizinghormone, vasopressin and vasopressin analogs, catalase, superoxidedismutase, interleukin-11, interferon, colony stimulating factor, tumornecrosis factor, tumor necrosis factor inhibitor, andmelanocyte-stimulating hormone. Interleukin eleven (IL-11) is athrombopoietic growth factor that directly stimulates the proliferationof hematopoietic stem cells and megakaryocyte progenitor cells andinduces megakaryocyte maturation resulting in increased plateletproduction (Oprelvekin®). In one preferred embodiment, the bioactiveagent is an Alzheimer antagonist.

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance epithelial permeation or paracellular transportcomprising γ-PGA or α-PGA (or other PGA derivatives, such asPGA-complexone conjugate that is negatively charged) and low molecularweight chitosan, wherein the chitosan dominates on a surface of thenanoparticles. Some aspects of the invention relate to an oral dose ofnanoparticles that effectively enhance epithelial permeation (e.g.,intestinal or blood brain paracellular transport) comprising a negativecomponent, such as γ-PGA, α-PGA, heparin, or heparan sulfate, in thecore, and low molecular weight chitosan dominating on the surface of thenanoparticles with positive surface charges. The core substrate may beselected from the group consisting of heparin, heparin analogs, lowmolecular weight heparin, glycosaminoglycans, and alginate, whereas thebioactive agent is selected from the group consisting of chondroitinsulfate, hyaluronic acid, growth factor and protein with apharmaceutically effective amount.

In a further embodiment, the nanoparticles comprise at least onebioactive agent, such as insulin, insulin analog, Alzheimer's diseaseantagonist, Parkinson's disease antagonist, or other protein/peptide.The bioactive agent for treating Alzheimer's disease may includememantine hydrochloride (Axura® by Merz Pharmaceuticals), donepezilhydrochloride (Aricept® by Eisai Co. Ltd.), rivastigmine tartrate(Exelon® by Novartis), galantamine hydrochloride (Reminyl® by Johnson &Johnson), or tacrine hydrochloride (Cognex® by Parke Davis). Examples ofinsulin or insulin analog products include, but not limited to, Humulin®(by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (by Aventis).

Some aspects of the invention provide a dose of nanoparticles thatenhance epithelial permeation, intestinal permeation, or blood brainparacellular transport. Each nanoparticle comprises three components;the first component of at least one bioactive agent, a second componentof low molecular weight chitosan that is positively charged and a thirdcomponent that is negatively charged, wherein the second componentdominates on a surface of the nanoparticle. In one embodiment, the thirdcomponent is γ-PGA, α-PGA, derivatives (such as PGA-complexone conjugateand the like), or salts of PGA, heparin or alginate. In anotherembodiment, the first component comprises insulin at a concentrationrange of 0.075 to 0.091 mg/ml, the second component at a concentrationrange of 0.67 to 0.83 mg/ml, and the third component comprises γ-PGA ata concentration range of 0.150 to 0.184 mg/ml. The at least onebioactive agent may comprise an antagonist for Alzheimer's disease orfor treatment of Alzheimer's disease selected from the group consistingof memantine hydrochloride, donepezil hydrochloride, rivastigminetartrate, galantamine hydrochloride, and tacrine hydrochloride. In afurther embodiment, the at least one bioactive agent is insulin orinsulin analog. In still another embodiment, the at least one bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, oxygen-enriching agent, oxygen-containing agent,calcitonin, vancomycin, and anti-inflammatory drugs.

Some aspects of the invention provide a dose of nanoparticles thatenhance permeation, wherein the nanoparticles are further encapsulatedin a capsule or hard-cap capsule. In one embodiment, the nanoparticlesare freeze-dried. In one embodiment, the interior surface of the capsuleis treated to be lipophilic or hydrophobic so to keep the enclosedingredients or nanoparticles intact or passive to the interior surface.In another embodiment, the interior surface or the exterior surface ofthe capsules is enteric-coated.

Some aspects of the invention provide a method of enhancing epithelialpermeation comprising administering a dose of nanoparticles, whereineach nanoparticle comprises a first component of at least one bioactiveagent, a second component of low molecular weight chitosan, and a thirdcomponent that is negatively charged, wherein the second componentdominates on a surface of the nanoparticle. In one embodiment, the stepof administering the dose of nanoparticles is via oral administrationfor enhancing epithelial permeation or intestinal paracellulartransport. In another embodiment, the step of administering the dose ofnanoparticles is via intravenous administration or injection to a bloodvessel for enhancing blood brain paracellular transport or reducing theblood-brain barrier (BBB). In another embodiment, the step ofadministering the nanoparticles is via subcutaneous injection,intramuscular injection, or intranasal spraying.

In one embodiment, the orally administered insulin-containingnanoparticles comprise an effective dosage amount of the insulin totreat the diabetes between about 5 units to 95 units insulin, preferablybetween about 15 units to 45 units, per kilogram body weight of theanimal subject. In a further embodiment, the insulin-containingnanoparticle comprises a trace amount of zinc or calcium, or is treatedwith enteric coating.

In one embodiment, the bioactive agent-containing nanoparticles furthercomprise at least one permeation enhancer, wherein the permeationenhancer may be selected from the group consisting of Ca²⁺ chelators,bile salts, anionic surfactants, medium-chain fatty acids, phosphateesters, and the like. In another embodiment, the nanoparticles and thepermeation enhancer are co-encapsulated in a capsule or are encapsulatedseparately in two sets of capsules for co-administration.

Some aspects of the invention provide a method of treating Alzheimer'sdiseases of an animal subject comprising intravenously administeringbioactive nanoparticles with an effective dosage to treat theAlzheimer's diseases, wherein the bioactive nanoparticles comprises apositively charged shell substrate, a negatively charged orsubstantially neutral-charged core substrate, and at least one bioactiveagent for treating Alzheimer's disease, wherein at least one bioactiveagent is selected from the group consisting of memantine hydrochloride,donepezil hydrochloride, rivastigmine tartrate, galantaminehydrochloride, and tacrine hydrochloride.

In one embodiment, the effective treatment of the Alzheimer's diseasescomprises administering at least one bioactive agent for treatingAlzheimer's diseases at about 10 mg to 40 mg per day over a period ofone month to one year or longer. In another embodiment, at least aportion of the shell substrate is crosslinked, preferably at a degree ofcrosslinking less than about 50%, or most preferably between about 1%and 20%.

One aspect of the invention provides a pharmaceutical composition ofnanoparticles, wherein the nanoparticles may be freeze-dried to formsolid dried nanoparticles. The dried nanoparticles may be loaded in acapsule (such as a two-part hard gelatin capsule) or a tablet, which maybe further enterically coated, for oral administration in an animalsubject. The freeze-dried nanoparticles can be rehydrated in a solutionor by contacting body fluid as to revert to wet nanoparticles havingpositive surface charge with substantially the properties of thepre-lyophilized nanoparticles. In one embodiment, nanoparticles may bemixed with trehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-dryingprocess. In one embodiment, the interior surface of the capsule istreated to be lipophilic or hydrophobic. In another embodiment, theexterior surface of the capsule is enteric-coated.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles that enhance epithelial permeation or paracellulartransport, each nanoparticle comprising a shell component and a corecomponent, wherein at least a portion of the shell component compriseschitosan and wherein the core component is comprised of MgSO₄, sodiumtripolyphosphate, at least one bioactive agent, and a negatively chargedcompound, wherein a substantial portion of the negatively chargedcompound is electrostatically bound to the chitosan.

Some aspects of the invention provide an orally deliverable capsule toan animal subject comprising: (a) an empty capsule; and (b) bioactivenanoparticles loaded within the empty capsule, wherein the nanoparticlescomprise a shell substrate of chitosan, a negatively charged orsubstantially neutral-charged core substrate, and (c) at least onebioactive agent. In one embodiment, the empty capsule comprises atwo-part hard gelatin capsule. In another embodiment, the capsule istreated with enteric coating. In one embodiment, the interior surface ofthe capsule may be treated with hydrophobic or enteric coating.

One object of the present invention is to provide a method ofmanufacturing the orally deliverable capsule, the method comprising thesteps of: (a) providing an empty capsule; (b) providing bioactivenanoparticles, wherein the nanoparticles comprise a shell substrate ofchitosan, a negatively charged or substantially zero-charged coresubstrate, and at least one bioactive agent; (c) freeze-drying thenanoparticles; and (d) filling the freeze-dried bioactive nanoparticlesinto the empty capsule, thereby producing an orally deliverable capsule.In one embodiment, the bioactive nanoparticles further comprise zinc,magnesium sulfate and TPP.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles for oral administration in an animal subject, thenanoparticles comprising a shell portion that is dominated by positivelycharged chitosan, a core portion that contains negatively chargedsubstrate, wherein the negatively charged substrate is at leastpartially neutralized with a portion of the positively charged chitosanin the core portion, and at least one bioactive agent loaded within thenanoparticles. In one embodiment, the bioactive agent is a non-insulinexenatide, a non-insulin pramlintide, GLP-1, GLP-1 analog, GLP-2, GLP-2analog, insulin, insulin analog, or combinations thereof. In oneembodiment, the nanoparticles are formed via a simple and mildionic-gelation method. Glucagon-like peptide-2 (GLP-2) is a recentlyidentified potent intestinotrophic factor. The effect of GLP-2 treatmenton intestinal epithelial barrier function in mice shows that theglucagon-like peptide-2 enhances intestinal epithelial barrier functionof both transcellular and paracellular pathways in the mouse.Nevertheless, glucagon-like peptide-2 reduces intestinal permeabilitybut does not modify the onset of Type 1 diabetes in the nonobesediabetic mouse.

In one embodiment, a surface of the nanoparticles of the pharmaceuticalcomposition of the present invention is characterized with a positivesurface charge, wherein the nanoparticles have a surface charge fromabout +15 mV to about +50 mV. In another embodiment, the nanoparticleshave a mean particle size between about 50 and 400 nanometers. In stillanother embodiment, at least a portion of the shell portion of thenanoparticles is crosslinked. In a further embodiment, the nanoparticlesare in a form of freeze-dried powder. In one embodiment, thenanoparticles of the pharmaceutical composition of the present inventionfurther comprise iron, zinc, calcium, magnesium sulfate and TPP.

Some aspects of the invention provide a method of delivering a bioactiveagent to blood circulation in an animal subject, comprising: (a)providing nanoparticles according to the pharmaceutical composition ofthe present invention, wherein the nanoparticles are formed via a simpleand mild ionic-gelation method; (b) administering the nanoparticlesorally toward an intestine of the animal subject; (c) urging thenanoparticles to be absorbed onto a surface of an epithelial membrane ofthe intestine; (d) permeating bioactive agent to pass through anepithelial barrier of the intestine; and (e) releasing the bioactiveagent into the blood circulation. In one embodiment, the bioactive agentis selected from the group consisting of exenatide, pramlintide,insulin, insulin analog, and combinations thereof.

Some aspects of the invention provide a method of delivering a bioactiveagent to an animal subject orally, the method comprising formulatingnanoparticles containing bioactive agent according to the principles ofthe present disclosure, wherein the nanoparticles are suspended inliquid. In one embodiment, the liquid with nanoparticles containingbioactive agent is served as a sport drink or energy drink. In oneembodiment, the bioactive agent is an oxygen-enriching agent oroxygen-containing agent (such as hemoglobin). In another embodiment, thebioactive agent is an energy-enhancing agent, such as CoQ₁₀. CoenzymeQ₁₀ (also known as ubiquinone, ubidecarenone, coenzyme Q, andabbreviated at times to CoQ₁₀, CoQ, Q10, or Q) is a benzoquinone, whereQ refers to the quinone chemical group, and 10 refers to the isoprenylchemical subunits. This oil-soluble vitamin-like substance is present inmost eukaryotic cells, primarily in the mitochondria. It is a componentof the electron transport chain and participates in aerobic cellularrespiration, generating energy in the form of ATP. Ninety-five percentof the human body's energy is generated this way. Therefore, thoseorgans with the highest energy requirements—such as the heart and theliver—have the highest CoQ₁₀ concentrations or requirement.

Some aspects of the invention provide a method of reducing inflammatoryresponse caused by tumor necrosis factor in an animal subject, themethod comprising orally administering nanoparticles composed of a TNFinhibitor, chitosan, and a core substrate of poly(glutamic acid) orheparin. In one embodiment, the TNF inhibitor is a monoclonal antibody.In another embodiment, the TNF inhibitor is infliximab or adalimumab. Inone embodiment, the TNF inhibitor is a circulating receptor fusionprotein. In another embodiment, the TNF inhibitor is etanercept.

In one embodiment, the chitosan of the nanoparticles has a molecularweight about 80 kDa or less. In another embodiment, the chitosan isN-trimethyl chitosan or chitosan derivatives, such as EDTA-chitosan. Instill another embodiment, the poly(glutamic acid) of the nanoparticlesis γ-PGA, α-PGA, and derivatives of PGA (such as PGA-complexoneconjugate), salts of PGA, or combinations thereof. In one embodiment,the nanoparticles have a mean particle size between about 50 and 400nanometers.

Some aspects of the invention relate to a method of treating infectionscaused by microorganisms in an animal subject, the method comprisingadministering nanoparticles composed of an antibiotic, chitosan, and acore portion of negatively charged substrate, wherein a surface of thenanoparticles is dominated by the chitosan.

In one embodiment, the antibiotic is selected from the group consistingof vancomycin, glycylcycline antibiotics (such as Tigecycline),lincosamide antibiotics (such as Lincomycin), beta-lactam antibiotic(such as Penicillin, Ampicillin, and Piperacillin), bacteriophages,antitumor antibiotics, and aminoglycoside antibiotics.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles, the nanoparticles consisting of a shell portion that isdominated by positively charged chitosan, a core portion that consistsof the positively charged chitosan, one negatively charged substrate, atleast one bioactive agent loaded within the nanoparticles, andoptionally a zero-charge compound, wherein the nanoparticles areenclosed within a physical structure to minimize direct exposure to anoutside environment or to isolate/seal the nanoparticles from an outsideenvironment. In one embodiment, the nanoparticles are loaded in acapsule. In another embodiment, the capsule is filled with amoisture-free gas, wherein the moisture-free gas is nitrogen, carbondioxide, or nitrogen-based inert gas.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles loaded with at least one bioactive agent, wherein thebioactive agent is protein or a peptide that is covalently attached withpolyethylene glycol polymer chains. In one embodiment, the bioactiveagent is an anti-diabetic drug that is covalently attached withpolyethylene glycol polymer chains.

Some aspects of the invention provide a method of treating diabetes in asubject, comprising administering bioactive nanoparticles to thesubject, wherein the nanoparticles comprise a shell portion that isdominated by positively charged chitosan, a core portion that containsnegatively charged substrate, wherein the negatively charged substrateis at least partially neutralized with a portion of the positivelycharged chitosan and at least one non-insulin anti-diabetic drug. In oneembodiment, the nanoparticles are encapsulated in capsules. In anotherembodiment, the capsules further comprise a second non-insulinanti-diabetic drug of the at least one non-insulin anti-diabetic drug.In one embodiment, the non-insulin anti-diabetic drug is metformin, aninsulin secretagogue, a GLP-1 analog, a DPP-4 inhibitor, or selectedfrom the group consisting of alpha-glucosidase inhibitors, amylinanalog, sodium-glucose co-transporter type 2 (SGLT2) inhibitors,benfluorex, and tolrestat. In another embodiment, the bioactivenanoparticles further comprise insulin or insulin analog. There areseveral reports suggesting that GLP-1 or GLP-1 analog has anappetite-suppressing action.

Some aspects of the invention provide administering bioactivenanoparticles to a subject with enhanced enzymatic resistance to thebioactive agent inside the bioactive nanoparticles, wherein thenanoparticles comprise a shell portion that is dominated by positivelycharged chitosan, a core portion that contains negatively chargedsubstrate, wherein the negatively charged substrate is at leastpartially neutralized with a portion of the positively charged chitosanand at least one enzyme-resistant agent. In one embodiment, theenzyme-resistant agent is complexone, such as diethylene triaminepentaacetic acid (DTPA) or ethylene diamine tetraacetic acid (EDTA),which may conjugate with the chitosan substrate or the PGA substrate inthe nanoparticle formulation.

Some aspects of the invention relate to a pharmaceutical composition ofnanoparticles, the nanoparticles comprising a shell portion that isdominated by positively charged chitosan, a core portion that comprisesone negatively charged substrate, wherein the substrate isPGA-complexone conjugate, wherein the negatively charged substrate is atleast partially neutralized with a portion of the positively chargedchitosan in the core portion, and at least one bioactive agent loadedwithin the nanoparticles. In one embodiment, the pharmaceuticalcomposition of nanoparticles further comprises a pharmaceuticallyacceptable carrier, diluent, or excipient.

In one embodiment, the nanoparticles are encapsulated in capsules,wherein the capsules further comprise at least a solubilizer, bubblingagent, dispersing agent, emulsifier, pharmacopoeial excipients or atleast one permeation enhancer. In another embodiment, the nanoparticlesare freeze-dried, thereby the nanoparticles being in a powder form.

In one preferred embodiment, the nanoparticles are encapsulated incapsules, wherein the capsules further comprise at least one permeationenhancer that is encapsulated in second nanoparticles, wherein thesecond nanoparticles may be formulated without any bioactive agent orwith a bioactive agent different from the first bioactive agent in thefirst afore-mentioned nanoparticles.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles, the nanoparticles consisting of a shell portion that isdominated by positively charged chitosan, a core portion that consistsof the positively charged chitosan, one negatively charged substrate, atleast one bioactive agent loaded within the nanoparticles, andoptionally a zero-charge compound, wherein the at least one bioactiveagent is an anti-hemophilic factor. In one embodiment, theanti-hemophilic factor is a recombinant human coagulation factor VIIa(rFVIIa) or a recombinant plasma/serum-free coagulation factor VIII. Inanother embodiment, the anti-hemophilic factor is a coagulation factorto treat the animal subject that lacks functional clotting factor VIII,factor IX, or factor XI. In another embodiment, the anti-hemophilicfactor is covalently attached with polyethylene glycol polymer chains.In still another embodiment, the anti-hemophilic factor is aglycoprotein procofactor.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional objects and features of the present invention will becomemore apparent and the disclosure itself will be best understood from thefollowing Detailed Description of the Exemplary Embodiments, when readwith reference to the accompanying drawings.

FIG. 1 shows GPC chromatograms of (a) standard-MW CS beforedepolymerization and the low-MW CS after depolymerization; (b) thepurified γ-PGA obtained from microbial fermentation.

FIG. 2 shows (a) FT-IR and (b) ¹H-NMR spectra of the purified γ-PGAobtained from microbial fermentation.

FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared CS-γ-PGAnanoparticles.

FIG. 4 shows (a) a TEM micrograph of the prepared CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) an AFM micrograph of the preparedCS-γ-PGA nanoparticles (0.01% γ-PGA:0.01% CS).

FIG. 5 shows changes in particle size and zeta potential of (a) theCS-γ-PGA nanoparticles (0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGAnanoparticles (0.10% γ-PGA:0.01% CS) during storage for up to 6 weeks.

FIG. 6 shows effects of the prepared CS-γ-PGA nanoparticles on the TEERvalues of Caco-2 cell monolayers.

FIG. 7 shows fluorescence images (taken by an inversed confocal laserscanning microscope) of 4 optical sections of a Caco-2 cell monolayerthat had been incubated with the fCS-γ-PGA nanoparticles with a positivesurface charge (0.10% γ-PGA:0.20% CS) for (a) 20 min and (b) 60 min.

FIG. 8 shows an illustrative protein transport mechanism through a celllayer, including transcellular transport and paracelluler transport.

FIGS. 9 A-C show a schematic illustration of a paracellular transportmechanism.

FIG. 10 shows an fCS-γ-PGA nanoparticle with FITC-labeled chitosanhaving positive surface charge.

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA.

FIG. 12 shows loading capacity and association efficiency of insulin innanoparticles of chitosan as reference.

FIG. 13 shows the stability of insulin-loaded nanoparticles.

FIG. 14 shows a representative in vitro study with insulin drug releaseprofile in a pH-adjusted solution.

FIG. 15 shows the effect of insulin of orally administeredinsulin-loaded nanoparticles on hypoglycemia in diabetic rats.

FIGS. 16 A-C show a proposed mechanism of nanoparticles released fromthe enteric coated capsules.

FIG. 17 shows the schematic illustration of insulin conjugated withhistidine and/or glutamic acid side groups of the γ-PGA via zinc.

FIG. 18 shows the schematic illustration of insulin conjugated with acarboxyl side group of the γ-PGA via zinc.

FIG. 19 shows the effect of orally administered insulin-loadednanoparticles on ‘glucose reduction %’ in diabetic rats, wherein thefreeze-dried nanoparticles were loaded in an enterically coated capsuleupon delivery.

FIG. 20 shows insulin-loaded nanoparticles with a core compositionconsisted of γ-PGA, MgSO₄, sodium tripolyphosphate (TPP), and insulin.

FIG. 21 shows an in vivo subcutaneous study using insulin injectablesand insulin-containing nanoparticles.

FIG. 22 shows experimental data on enzyme inhibition study with(γ-PGA)-DTPA conjugate.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The preferred embodiments of the present invention described belowrelate particularly to the preparation of nanoparticles composed ofchitosan/poly-glutamic acid/insulin and their permeability to enhancethe intestinal or blood brain paracellular permeation by opening thetight junctions between epithelial cells. While the description setsforth various embodiment specific details, it should be understood thatthe description is illustrative only and should not be construed in anyway as limiting the invention. Furthermore, various applications of theinvention, and modifications thereto, which may occur to those who areskilled in the art, are also encompassed by the general conceptsdescribed below.

“Bioactive agent” herein is meant to include any agent that may affectthe recipient (an animal subject) after being administered physically,physiologically, mentally, biochemically, biologically, or other bodilyfunctions in a positive or negative manners. The ‘bioactive agent’ mayinclude, but not limited to, drugs, protein, peptides, siRNA, enzymes,supplemental nutrients, vitamins, other active agents. A pegylatedbioactive agent is a bioactive agent with covalent attachment ofpolyethylene glycol polymer chains.

γ-PGA is a naturally occurring anionic homo-polyamide that is made ofL-glutamic acid units connected by amide linkages between α-amino andγ-carboxylic acid groups (Crit. Rev. Biotechnol. 2001; 21:219-232). Itis an exocellular polymer of certain Bacillus species that is producedwithin cells via the TCA cycle and is freely excreted into thefermentation broth. Its exact biological role is not fully known,although it is likely that γ-PGA is linked to increasing the survival ofproducing strains when exposed to environmental stresses. Because of itswater-solubility, biodegradability, edibility, and non-toxicity towardhumans and the environment, several applications of γ-PGA in food,cosmetics, medicine, and water treatment have been investigated in thepast few years.

Example No. 1 Materials and Methods of Nanoparticles Preparation

CS (MW ˜2.8×10⁵) with a degree of deacetylation of approximately 85% wasacquired from Challenge Bioproducts Co. (Taichung, Taiwan). Acetic acid,cellulase (1.92 units/mg), fluorescein isothiocyanate (FITC), phosphatebuffered saline (PBS), periodic acid, sodium acetate, formaldehyde,bismuth subnitrate, and Hanks' balanced salt solution (HBSS) werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absoluteanhydrous and potassium sodium tartrate were obtained from Merck(Darmstadt, Germany). Non-essential amino acid (NEAA) solution, fetalbovine serum (FBS), gentamicin and trypsin-EDTA were acquired from Gibco(Grand Island, N.Y.). Eagle's minimal essential medium (MEM) waspurchased from Bio West (Nuaille, France). All other chemicals andreagents used were of analytical grade.

Example No. 2 Depolymerization of CS by Enzymatic Hydrolysis

Regular CS was treated with enzyme (cellulase) to produce low-MW CSaccording to a method described by Qin et al. with some modifications(Food Chem. 2004; 84:107-115). A solution of CS (20 g/l) was prepared bydissolving CS in 2% acetic acid. Care was taken to ensure the totalsolubility of CS. Then, the CS solution was introduced into a vessel andadjusted to the desired pH 5.0 with 2N aqueous NaOH. Subsequently,cellulase (0.1 g) was added into the CS solution (100 ml) andcontinuously stirred at 37° C. for 12 hours. Afterward, thedepolymerized CS was precipitated with aqueous NaOH at pH 7.0-7.2 andthe precipitated CS was washed three times with deionized water. Theresulting low-MW CS was lyophilized in a freeze dryer (Eyela Co. Ltd,Tokyo, Japan).

The average molecular weight of the depolymerized CS was determined by agel permeation chromatography (GPC) system equipped with a series of PLaquagel-OH columns (one Guard 8 μm, 50×7.5 mm and two MIXED 8 μm,300×7.5 mm, PL Laboratories, UK) and a refractive index (RI) detector(RI2000-F, SFD, Torrance, Calif.). Polysaccharide standards (molecularweights range from 180 to 788,000, Polymer Laboratories, UK) were usedto construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.5M NaNO₃ and was brought to a pH of 2.0. The flow rate ofthe mobile phase was 1.0 ml/min, and the columns and the RI detectorcell were maintained at 30° C.

Factors limiting applications of most commercially available CSs aretheir high molecular weight and corresponding high viscosity and poorsolubility at physiological pH ranges. Low-MW CS overcomes theselimitations and hence finds much wider applications in diversifiedfields. It was suggested that low-MW CS be used as a parenteral drugcarrier due to its lower antigen effect (Eur. J. Pharm. Biopharm. 2004;57:101-105). Low-MW CS was used as a non-viral gene delivery system andshowed promising results (Int. J. Pharm. 1999; 178:231-243). Otherstudies based on animal testing showed the possibilities of low-MW CSfor treatment of type 2 diabetes and gastric ulcer (Biol. Pharm. Bull.2002; 25:188-192). Several hydrolytic enzymes such as lysozyme,pectinase, cellulase, bromelain, hemicellulase, lipase, papain and thelike can be used to depolymerize CS (Biochim. Biophys. Acta 1996;1291:5-15; Biochem. Eng. J. 2001; 7:85-88; Carbohydr. Res. 1992;237:325-332).

FIG. 1 a shows GPC chromatograms of both standard-MW (also known asregular-MW) and low-MW CS. It is known that cellulase catalyzes thecleavage of the glycosidic linkage in CS (Food Chem. 2004; 84:107-115).The low-MW CS used in the study was obtained by precipitating thedepolymerized CS solution with aqueous NaOH at pH 7.0-7.2. This low-MWCS had a MW of about 50 kDa (FIG. 1 a). In a preferred embodiment, thelow molecular weight chitosan has a molecular weight of less than about40 kDa, but above 10 kDa. Other forms of chitosan may also beapplicable, including chitin, chitosan oligosaccharides, and derivativesthereof.

It was observed that the obtained low-MW CS can be readily dissolved inan aqueous solution at pH 6.0, while that before depolymerization needsto be dissolved in an acetic acid solution with a pH value about 4.0.Additionally, it was found that with the low-MW CS, the preparednanoparticles had a significantly smaller size with a narrowerdistribution than their counterparts prepared with the high-MW (alsoknown as standard-MW) CS (before depolymerization), due to its lowerviscosity. As an example, upon adding a 0.10% γ-PGA aqueous solutioninto a 0.20% high-MW CS solution (viscosity 5.73±0.08 cp, measured by aviscometer), the mean particle size of the prepared nanoparticles was878.3±28.4 nm with a polydispersity index of 1.0, whereas adding a 0.10%γ-PGA aqueous solution into the low-MW CS solution (viscosity 1.29±0.02cp) formed nanoparticles with a mean particle size of 218.1±4.1 nm witha polydispersity index of 0.3 (n=5).

The purified γ-PGA used in forming nanoparticles of the presentinvention was analyzed by GPC, ¹H-NMR, and FT-IR. As analyzed by GPC(FIG. 1 b), the purified γ-PGA had a MW of about 160 kDa. In the FT-IRspectrum (FIG. 2 a), a characteristic peak at 1615 cm⁻¹ for theassociated carboxylic acid salt (—COO⁻ antisymmetric stretch) on γ-PGAwas observed. The characteristic absorption due to C═O in secondaryamides (amide I band) was overlapped by the characteristic peak of—COO⁻. Additionally, the characteristic peak observed at 3400 cm⁻ wasthe N—H stretch of γ-PGA. In the ¹H-NMR spectrum (FIG. 2 b), six chiefsignals were observed at 1.73 and 1.94 ppm (β-CH₂), 2.19 ppm (γ-CH₂),4.14 ppm (α-CH), 8.15 ppm (amide), and 12.58 ppm (COOH). These resultsindicated that the observed FT-IR and ¹H-NMR spectra correspond well tothose expected for γ-PGA. Additionally, the fermented product afterpurification showed no detected macromolecular impurities by the ¹H-NMRanalysis, suggesting that the obtained white power of γ-PGA is highlypure.

Example No. 3 Preparation of the CS-γ-PGA Nanoparticles

Nanoparticles were obtained upon addition of γ-PGA aqueous solution (pH7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at varying concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% byw/v) under magnetic stirring at room temperature. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatantswere discarded and nanoparticles were resuspended in deionized water forfurther studies. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge and a narrow polydispersity index. FT-IRwas used to analyze peak variations of amino groups of low-MW CS andcarboxylic acid salts of γ-PGA in the CS-γ-PGA nanoparticles.

As stated, nanoparticles were obtained instantaneously upon the additionof a γ-PGA aqueous solution (pH 7.4) into a low-MW CS aqueous solution(pH 6.0) under magnetic stirring at room temperature. FIG. 3 shows theFT-IR spectra of the low-MW CS and the CS-γ-PGA nanoparticles. As shownin the spectrum of CS, the characteristic peak observed at 1563 cm⁻ wasthe protonated amino group (—NH₃ ⁺ deformation) on CS. In the spectrumof CS-γ-PGA complex, the characteristic peak at 1615 cm⁻ for —COO⁻ onγ-PGA disappeared and a new peak at 1586 cm⁻ appeared, while thecharacteristic peak of —NH₃ ⁺ deformation on CS at 1563 cm⁻ shifted to1555 cm⁻¹. These observations are attributed to the electrostaticinteraction between the negatively charged carboxylic acid salts (—COO⁻)on γ-PGA and the positively charged amino groups (—NH₃ ⁺) on CS (Int. J.Pharm. 2003; 250:215-226). The electrostatic interaction between the twopolyelectrolytes (γ-PGA and CS) instantaneously induced the formation oflong hydrophobic segments (or at least segments with a high density ofneutral ion-pairs), and thus resulted in highly neutralized complexesthat segregated into colloidal nanoparticles (Langmuir. 2004;20:7766-7778).

The particle sizes and the zeta potential values of the CS-γ-PGAnanoparticles, prepared at varying concentrations of the γ-PGA and CS,were determined and the results are shown in Tables 1a and 1b. It wasfound that the particle size and the zeta potential value of theprepared nanoparticles were mainly determined by the relative amount ofthe local concentration of the γ-PGA in the added solution to thesurrounding concentration of CS in the sink solution. At a fixedconcentration of CS, an increase in the γ-PGA concentration allowedγ-PGA molecules to interact with more CS molecules, and thus formed alarger size of nanoparticles (Table 1a, p<0.05). When the amount of CSmolecules exceeded that of local γ-PGA molecules, some of the excess CSmolecules were entangled onto the surfaces of CS-γ-PGA nanoparticles.

Thus, the resulting nanoparticles may display a structure of a neutralpolyelectrolyte-complex core surrounded by a positively charged CS shell(Table 1b) ensuring a colloidal stabilization (Langmuir. 2004;20:7766-7778). In contrast, as the amount of local γ-PGA moleculessufficiently exceeded that of surrounding CS molecules, the formednanoparticles had γ-PGA exposed on the surfaces and thus had a negativecharge of zeta potential. Therefore, the particle size and the zetapotential value of the prepared CS-γ-PGA nanoparticles can be controlledby their constituted compositions. The results obtained by the TEM andAFM examinations showed that the morphology of the preparednanoparticles was spherical in shape with a smooth surface (FIGS. 4 aand 4 b). Some aspects of the invention relate to nanoparticles having amean nanoparticle size between about 50 and 400

TABLE 1a Effects of concentrations of γ-PGA and CS on the particle sizesof the prepared CS-γ-PGA nanoparticles Mean Particle Size (nm, n = 5) CSγ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))  79.0 ± 3.0 103.1 ±4.6  96.7 ± 1.9 103.6 ± 1.9 140.5 ± 2.0 0.05% 157.4 ± 1.7 120.8 ± 3.9144.5 ± 2.4 106.2 ± 3.8 165.4 ± 1.7 0.10% 202.2 ± 3.1 232.6 ± 1.2 161.0± 1.8 143.7 ± 2.7 218.1 ± 4.1 0.15% 277.7 ± 3.2 264.9 ± 2.1 188.6 ± 2.9178.0 ± 2.2 301.1 ± 6.4 0.20% 284.1 ± 2.1 402.2 ± 4.0 ▴ 225.5 ± 3.1365.5 ± 5.1 ^(a))concentration of CS (by w/v) ^(b))concentration ofγ-PGA (by w/v) ▴ precipitation of aggregates was observed

TABLE 1b Effects of concentrations of γ-PGA and CS on the zeta potentialvalues of the prepared CS-γ-PGA nanoparticles. Zeta Potential (mV, n =5) CS γ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))   15.4 ± 0.322.8 ± 0.5 19.8 ± 1.5 16.5 ± 1.4 17.2 ± 1.6 0.05% −32.7 ± 0.7 23.7 ± 1.727.6 ± 0.7 20.3 ± 0.8 19.2 ± 0.6 0.10% −33.1 ± 1.3 21.1 ± 1.6 20.3 ± 1.123.6 ± 0.9 24.7 ± 1.2 0.15% −33.2 ± 2.1 −21.9 ± 2.0   19.2 ± 0.4 16.9 ±1.7 19.8 ± 0.3 0.20% −34.5 ± 0.5 −34.6 ± 0.3   ▴ 14.6 ± 0.7 16.3 ± 0.7^(a))concentration of CS (by w/v) ^(b))concentration of γ-PGA (by w/v) ▴precipitation of aggregates was observednanometers, preferably between about 100 and 300 nanometers, and mostpreferably between about 100 and 200 nanometers.

The morphology of the nanoparticles is spherical in shape with a smoothsurface at any pH between 2.5 and 6.6. In one embodiment, the stabilityof the nanoparticles of the present invention at a low pH around 2.5enables the nanoparticles to be intact when exposed to the acidic mediumin the stomach.

Two representative groups of the prepared nanoparticles were selectedfor the stability study: one with a positive surface charge (0.10%γ-PGA:0.20% CS) and the other with a negative surface charge (0.10%γ-PGA:0.01% CS). FIG. 5 shows changes in particle size (▪, meandiameter) and zeta potential (●) of (a) the CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGA nanoparticles (0.10%γ-PGA:0.01% CS) during storage up to 6 weeks. It was found that neitheraggregation nor precipitation of nanoparticles was observed duringstorage up to 6 weeks, as a result of the electrostatic repulsionbetween the positively charged CS-γ-PGA nanoparticles (for the formergroup) and the negatively charged CS-γ-PGA nanoparticles (for the lattergroup).

Additionally, changes in particle size and zeta potential of thenanoparticles were minimal for both studied groups (FIGS. 5 a and 5 b).These results demonstrated that the prepared nanoparticles suspended indeionized water were stable during storage.

In a further study, NPs were self-assembled instantaneously uponaddition of an aqueous γ-PGA into an aqueous TMC(N-trimethyl chitosan)having a TMC/γ-PGA weight ratio of 6:1 under magnetic stirring at roomtemperature. Other chitosan derivative, such as mono-N-carboxymethylchitosan (MCC), has also been useful in self-assembled nanoparticleformation. The chemical formulas of chitosan, N-trimethyl chitosan, andMCC are shown below:

The amount of positively charged TMC significantly exceeded that ofnegatively charged γ-PGA; some of excessive TMC molecules were entangledonto the surfaces of NPs, thus displaying a positive surface charge(Table 2). The degree of quaternization on TMC had little effects on themean particle size and zeta potential of NPs.

TABLE 2 Mean particle sizes, zeta potential values and polydispersityindices of nanoparticles (NPs) self-assembled by TMC polymers withdifferent degrees of quaternization and γ-PGA (n = 5 batches). TMC:N-trimethyl chitosan; CS: chitosan; γ-PGA: poly(γ-glutamic acid). MeanParticle Size Zeta Potential Polydispersity (nm) (mV) Index CS/γ-PGA NPs104.1 ± 1.2 36.2 ± 2.5 0.11 ± 0.02 TMC25/γ-PGA NPs 101.3 ± 3.1 30.9 ±2.1 0.13 ± 0.04 TMC40/γ-PGA NPs 106.3 ± 2.3 32.3 ± 2.1 0.15 ± 0.14TMC55/γ-PGA NPs 114.6 ± 2.3 30.6 ± 3.8 0.12 ± 0.03

Example No. 4 Caco-2 Cell Cultures and TEER Measurements

Caco-2 cells were seeded on the tissue-culture-treated polycarbonatefilters (diameter 24.5 mm, growth area 4.7 cm²) in Costar Transwell 6wells/plates (Corning Costar Corp., NY) at a seeding density of 3×10⁵cells/insert. MEM (pH 7.4) supplemented with 20% FBS, 1% NEAA, and 40μg/ml antibiotic-gentamicin was used as the culture medium, and added toboth the donor and acceptor compartments. The medium was replaced every48 hours for the first 6 days and every 24 hours thereafter. Thecultures were kept in an atmosphere of 95% air and 5% CO₂ at 37° C. andwere used for the paracellular transport experiments 18-21 days afterseeding (TEER values in the range of 600-800 Ωcm²).

The intercellular tight junction is one of the major barriers to theparacellular transport of macromolecules (J. Control. Release 1996;39:131-138; J. Control. Release 1998; 51:35-46). Trans-epithelial iontransport is contemplated to be a good indication of the tightness ofthe junctions between cells and therefore evaluated by measuring TEER ofCaco-2 cell monolayers in the study. It was reported that themeasurement of TEER can be used to predict the paracellular transport ofhydrophilic molecules (Eur. J. Pharm. Biopharm. 2004; 58:225-235). Whenthe tight junctions open, the TEER value will be reduced due to thewater and ion passage through the paracellular route. Caco-2 cellmonolayers have been widely used as an in vitro model to evaluate theintestinal paracellular permeability of macromolecules.

Effects of the prepared CS-γ-PGA nanoparticles on the TEER values ofCaco-2 cell monolayers are shown in FIG. 6. As shown, the preparednanoparticles with a positive surface charge (CS dominated on thesurface, 0.01% γ-PGA:0.05% CS, 0.10% γ-PGA:0.2% CS, and 0.20%γ-PGA:0.20% CS) were able to reduce the values of TEER of Caco-2 cellmonolayers significantly (p<0.05). After a 2-hour incubation with thesenanoparticles, the TEER values of Caco-2 cell monolayers were reduced toabout 50% of their initial values as compared to the control group(without addition of nanoparticles in the transport media). Thisindicated that the nanoparticles with CS dominated on the surfaces couldeffectively open or loosen the tight junctions between Caco-2 cells,resulting in a decrease in the TEER values. It was reported thatinteraction of the positively charged amino groups of CS with thenegatively charged sites on cell surfaces and tight junctions induces aredistribution of F-actin and the tight junction's protein ZO-1, whichaccompanies the increased paracellular permeability (Drug Deliv. Rev.2001; 50:S91-S101). It is suggested that an interaction between chitosanand the tight junction protein ZO-1, leads to its translocation to thecytoskeleton.

After removal of the incubated nanoparticles, a gradual increase in TEERvalues was noticed. This phenomenon indicated that the intercellulartight junctions of Caco-2 cell monolayers started to recover gradually;however, the TEER values did not recover to their initial values (FIG.6). Kotzé et al. reported that complete removal of a CS-derived polymer,without damaging the cultured cells, was difficult due to the highlyadhesive feature of CS (Pharm. Res. 1997; 14:1197-1202). This might bethe reason why the TEER values did not recover to their initial values.In contrast, the TEER values of Caco-2 cell monolayers incubated withthe nanoparticles with a negative surface charge (γ-PGA dominated on thesurface, 0.10% γ-PGA:0.01% CS and 0.20% γ-PGA:0.01% CS, FIG. 6) showedno significant differences as compared to the control group (p>0.05).This indicated that γ-PGA does not have any effects on the opening ofthe intercellular tight junctions.

FIG. 8 shows an illustrative protein transport mechanism through acellular layer, including transcellular transport and paracellulertransport. FIG. 9 shows a schematic illustration of a paracellulartransport mechanism. The transcellular protein or peptide transport maybe either an active transport or a passive transport mode whereas theparacellular transport is basically a passive mode. Ward et al. reportedand reviewed current knowledge regarding the physiological regulation oftight junctions and paracellular permeability (PSTT 2000; 3:346-358).Chitosan as nanoparticle vehicles for oral delivery of protein drugsavoids the enzymatic inactivation in the gastrointestinal conduit. Thechitosan component of the present nanoparticles has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells; that is, loosening the tightness ofthe tight junctions.

FIG. 9(A) shows that after feeding nanoparticles (NPs) orally, NPsadhere and infiltrate into the mucus layer of the epithelial cells. FIG.9(B) illustrates that the infiltrated NPs transiently and reversiblyloosen tight junctions (TJs) while becoming unstable and disintegratedto release insulin or another entrapped agent. FIG. 9( c) shows that thereleased insulin or agent permeates through the paracellular pathwayinto the blood stream. Chitosan (CS), a nontoxic, soft-tissuecompatible, cationic polysaccharide has special features of adhering tothe mucosal surface; CS is able to transiently and reversiblywiden/loosen TJs between epithelial cells. The TJ width in the smallintestine has been demonstrated to be less than 1 nm. It is also knownthat TJs ‘opened’ by absorption enhancers are less than 20 nm wide(Nanotechnology 2007; 18:1-11). The term “opened” herein means that anysubstance less than 20 nm in the close-proximity might have the chanceto pass through. TJs are the principal barrier to passive movement offluid, electrolytes, macromolecules and cells through the paracellularpathway.

It was suggested that the electrostatic interaction between thepositively charged CS and the negatively charged sites of ZO-1 proteinson cell surfaces at TJ induces a redistribution of cellular F-actin aswell as ZO-1's translocation to the cytoskeleton, resulting in anincrease in permeability. As evidenced in FIG. 9, after adhering andinfiltrating into the mucus layer of the duodenum, the orallyadministered nanoparticles may degrade due to the presence of distinctdigestive enzymes in the intestinal fluids. Additionally, the pHenvironment may become neutral while the nanoparticles were infiltratinginto the mucosa layer and approaching the intestinal epithelial cells.This further leads to the collapse of nanoparticles due to the change inthe exposed pH environment. The dissociated CS from thedegraded/collapsed nanoparticles was then able to interact and modulatethe function of ZO-1 proteins between epithelial cells (Nanotechnology2007; 18:1-11). ZO-1 proteins are thought to be a linkage moleculebetween occludin and F-actin cytoskeleton as well as play importantroles in the rearrangement of cell-cell contacts at TJs.

Example No. 5 fCS-γ-PGA Nanoparticle Preparation and CLSM Visualization

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles (FIG. 10)were prepared for the confocal laser scanning microscopy (CLSM) study.The nanoparticles of the present invention display a structure of aneutral polyelectrolyte-complex core surrounded by a positively chargedchitosan shell. Synthesis of the FITC-labeled low-MW CS (fCS) was basedon the reaction between the isothiocyanate group of FITC and the primaryamino groups of CS as reported in the literature (Pharm. Res. 2003;20:1812-1819). Briefly, 100 mg of FITC in 150 ml of dehydrated methanolwere added to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hoursof reaction in the dark at ambient conditions, fCS was precipitated byraising the pH to about 8-9 with 0.5M NaOH. To remove the unconjugatedFITC, the precipitate was subjected to repeated cycles of washing andcentrifugation (40,000×g for 10 min) until no fluorescence was detectedin the supernatant. The fCS dissolved in 80 ml of 0.1M acetic acid, thendialyzed for 3 days in the dark against 5 liters of distilled water,with the water replaced on a daily basis. The resulting fCS waslyophilized in a freeze dryer. The fCS-γ-PGA nanoparticles were preparedas per the procedure described in Example No. 3.

Afterward, the transport medium containing fCS-γ-PGA nanoparticles (0.2mg/ml) was introduced into the donor compartment of Caco-2 cells, whichwere pre-cultured on the transwell for 18-21 days. The experimentaltemperature was maintained at 37° C. by a temperature control system(DH-35 Culture Dish Heater, Warner Instruments Inc., Hamden, Conn.).After incubation at specific time intervals, test samples wereaspirated. The cells were then washed twice with pre-warmed PBS solutionbefore they were fixed in 3.7% paraformaldehyde (Pharm. Res. 2003;20:1812-1819). Cells were examined under an inversed CLSM (TCS SL,Leica, Germany). The fluorescence images were observed using an argonlaser (excitation at 488 nm, emission collected at a range of 510-540nm).

A CLSM was used to visualize the transport of the fluorescence-labeledCS-γ-PGA (fCS-γ-PGA) nanoparticles across the Caco-2 cell monolayers.This non-invasive method allows for optical sectioning and imaging ofthe transport pathways across the Caco-2 cell monolayers, withoutdisrupting their structures (J. Control. Release 1996; 39:131-138).FIGS. 7 a and 7 b show the fluorescence images of 4 optical sections ofa Caco-2 cell monolayer that had been incubated with the fCS-γ-PGAnanoparticles having a positive surface charge (0.10% γ-PGA:0.20% CS,zeta potential: about 21 mV) for 20 and 60 min, respectively. As shown,after 20 min of incubation with the nanoparticles, intense fluorescencesignals at intercellular spaces were observed at depths of 0 and 5 μmfrom the apical (upper) surface of the cell monolayer. The intensity offluorescence became weaker at levels deeper than 10 μm from the apicalsurface of the cell monolayer and was almost absent at depths≧15 μm(FIG. 7 a).

After 60 minutes of incubation with the nanoparticles, the intensity ofthe fluorescence observed at intercellular spaces was stronger andappeared at a deeper level than those observed at 20 min afterincubation. These observations correlated with our TEER results,confirming that the nanoparticles with a positive surface charge (CSdominated on the surface) were able to open the tight junctions betweenCaco-2 cells and allow transport of the nanoparticles by passivediffusion via the paracellular pathways.

Example No. 6 In Vivo Study with Fluorescence-Labeled Nanoparticles

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles wereprepared for the confocal laser scanning microscopy (CLSM) study. Afterfeeding rats with fCS-γ-PGA nanoparticles, the rats were sacrificed at apre-determined time and the intestine isolated for CLSM examination. Thefluorescence images of the nanoparticles that showed penetration throughthe mouse intestine at appropriate time and at various depths from theinner surface toward the exterior surface of the intestine, includingduodenum, jejunum, and ileum were clearly observed by CLSM.

Example No. 7 Insulin Loading Capacity in Nanoparticles

Fluorescence (FITC)-labeled γ-PGA was added into the chitosan solutionto prepare fluorescence (FITC)-labeled, insulin-loaded CS-γ-PGAnanoparticles for in vivo animal study with confocal laser scanningmicroscopy (CLSM) assessment and bioactivity analysis. Theinsulin-loaded CS-γ PGA nanoparticles are prepared by using theionic-gelation method upon addition of insulin mixed with γ-PGA solutioninto CS solution, followed by magnetic stirring in a container.

Model insulin used in the experiment and disclosed herein is obtainedfrom bovine pancreas (Sigma-Aldrich, St. Louis, Mo.), having a molecularformula of C₂₅₄H₃₇₇N₆₅O₇₅S₆ with a molecular weight of about 5733.5 andan activity of ≧27 USP units/mg. The insulin contains a two-chainpolypeptide hormone produced by the β-cells of pancreatic islets. The αand β chains are joined by two interchain disulfide bonds. Insulinregulates the cellular uptake, utilization, and storage of glucose,amino acids, and fatty acids as well as inhibits the breakdown ofglycogen, protein, and fat. The insulin from Sigma-Aldrich containsabout 0.5% zinc. Separately, insulin can be obtained from other sources,such as a human insulin solution that is chemically defined, recombinantfrom Saccharomyces cerevisiae. Some aspects of the invention relate tonanoparticles with insulin in the core, wherein the insulin may containintermediate-acting, regular insulin, rapid-acting insulin,sustained-acting insulin that provides slower onset and longer durationof activity than regular insulin, or combinations thereof.

Examples of insulin or insulin analog products include, but not limitedto, Humulin® (by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (byAventis), and Novolog® Mix70/30 (by Novo Nordisk). Humalog (insulinlispro, rDNA origin) is a human insulin analog that is a rapid-acting,parenteral blood glucose-lowering agent. Chemically, it is Lys(B28),Pro(B29) human insulin analog, created when the amino acids at positions28 and 29 on the insulin B-chain are reversed. Humalog is synthesizedwith a special non-pathogenic laboratory strain of Escherichia colibacteria that has been genetically altered by the addition of the genefor insulin lispro. Humalog has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆and a molecular weight of 5808, identical to that of human insulin. Thevials and cartridges contain a sterile solution of Humalog for use as aninjection. Humalog injection consists of zinc-insulin lispro crystalsdissolved in a clear aqueous fluid. Each milliliter of Humalog injectioncontains insulin lispro 100 Units, 16 mg glycerin, 1.88 mg dibasicsodium phosphate, 3.15 mg m-cresol, zinc oxide content adjusted toprovide 0.0197 mg zinc ion, trace amounts of phenol, and water forinjection. Insulin lispro has a pH of 7.0-7.8. Hydrochloric acid 10%and/or sodium hydroxide 10% may be added to adjust pH.

Humulin is used by more than 4 million people with diabetes around theworld every day. Despite its name, this insulin does not come from humanbeings. It is identical in chemical structure to human insulin and ismanufactured in a factory using a chemical process called recombinantDNA technology. Humulin L is an amorphous and crystalline suspension ofhuman insulin with a slower onset and a longer duration of activity (upto 24 hours) than regular insulin. Humulin U is a crystalline suspensionof human insulin with zinc providing a slower onset and a longer andless intense duration of activity (up to 28 hours) compared to regularinsulin or the intermediate-acting insulins (NPH and Lente).

LANTUS® (insulin glargine [rDNA origin] injection) is a sterile solutionof insulin glargine for use as an injection. Insulin glargine is arecombinant human insulin analog that is a long-acting (up to 24-hourduration of action), parenteral blood-glucose-lowering agent. LANTUS isproduced by recombinant DNA technology utilizing a non-pathogeniclaboratory strain of Escherichia coli (K12) as the production organism.Insulin glargine differs from human insulin in that the amino acidasparagine at position A21 is replaced by glycine and two arginines areadded to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ with a molecular weight of 6063.

LANTUS consists of insulin glargine dissolved in a clear aqueous fluid.Each milliliter of LANTUS (insulin glargine injection) contains 100 IU(3.6378 mg) insulin glargine. Inactive ingredients for the 10 mL vialare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, 20 mcg polysorbate20, and water for injection. Inactive ingredients for the 3 mL cartridgeare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, and water forinjection. In 2006, there were 11.4 million prescriptions of Lantus inthe U.S. for basal insulin maintenance.

Novolog® Mix70/30 (70% insulin aspart protamine suspension and 30%insulin aspart injection [rDNA origin]) is a human insulin analogsuspension. Novolog® Mix70/30 is a blood glucose-lowering agent with arapid onset and an intermediate duration of action. Insulin aspart ishomologous with regular human insulin with the exception of a singlesubstitution of the amino acid praline by aspartic acid in position B28,and is produced by recombinant DNA technology utilizing Saccharomycescerevisiae as the production organism. Insulin aspart (Novolog) has theempirical formula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of 5826.Novolog® Mix70/30 is a uniform, white sterile suspension that containszinc 19.6 μg/ml and other components.

The nanoparticles with two insulin concentrations are prepared at achitosan to γ-PGA ratio of 0.75 mg/ml to 0.167 mg/ml. Their particlesize and zeta potential are shown in Table 3 below.

TABLE 3 Insulin Conc. Mean Particle Size Polydispersity Index ZetaPotential (mg/ml) (n = 5) (nm) (PI) (mV) 0*   145.6 ± 1.9 0.14 ± 0.01+32.11 ± 1.61 0.042 185.1 ± 5.6 0.31 ± 0.05 +29.91 ± 1.02 0.083 198.4 ±6.2 0.30 ± 0.09 +27.83 ± 1.22 *control reference without insulin

Further, their association efficiency of insulin and loading capacity ofinsulin are analyzed, calculated and shown in FIGS. 11 and 12, accordingto the following formula:

${{Insulin}\mspace{14mu}{Association}\mspace{14mu}{{Efficiency}\left( {{LE}\%} \right)}} = {\frac{\left( {{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} - {{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}} \right)}{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{unsulin}} \times 100\%}$${{Loading}\mspace{14mu}{{Capacity}({LC})}} = {\frac{\left( {{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} - {{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}} \right)}{{Weight}\mspace{14mu}{of}\mspace{14mu}{recovered}\mspace{14mu}{particles}} \times 100\%}$

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA, whereas FIG. 12 shows loadingcapacity and association efficiency of insulin in nanoparticles ofchitosan alone (in absence of γ-PGA) as reference. The data clearlydemonstrates that both the insulin loading capacity and insulinassociation efficiency are statistically higher for the nanoparticleswith γ-PGA in the core. The LE (40-55%) and LC (5.0˜14.0%) of insulinfor CS-γ PGA nanoparticles were obtained by using the ionic-gelationmethod upon addition of insulin mixed with γ-PGA solution into CSsolution, followed by magnetic stirring for nanoparticle separation.

In certain follow-up experiments, nanoparticles having a pharmaceuticalcomposition have been successfully prepared with a negatively chargedcomponent comprised of γ-PGA, α-PGA, PGA derivatives, salts of PGA,heparin or heparin analog, glycosaminoglycans, or alginate. The PGAderivatives of the present invention may include, but are not limitedto, poly-γ-glutamic acid, poly-α-glutamic acid, poly-L-glutamic acid(manufactured by Sigma-Aldrich, St. Louis, Mo.), poly-D-glutamic acid,poly-L-α-glutamic acid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid,and PEG or PHEG derivatives of polyglutamic acid, salts of theabove-cited PGAs, and the like. Some aspects of the invention relate tonanoparticles comprising a shell component and a core component, whereinat least a portion of the shell component comprises chitosan and whereinthe core component is comprised of a negatively charged compound that isconjugated to chitosan, and a bioactive agent. Some aspects of theinvention relate to an oral dose of nanoparticles that effectivelyenhance epithelial permeation (such as intestinal or blood brainparacellular transport) comprising a negative component (such as γ-PGA,α-PGA, PGA derivatives, heparin, or alginate) in the core and lowmolecular weight chitosan, wherein the chitosan dominates on a surfaceof the nanoparticles with positive charges.

Some aspects of the invention relate to a dose of nanoparticles thateffectively enhance epithelial permeation, intestinal transport or bloodbrain paracellular transport comprising a polyanionic component (such asγ-PGA, α-PGA, PGA derivatives, heparin, heparin analogs, low molecularweight heparin, glycosaminoglycans, or alginate) in the core and lowmolecular weight chitosan in the shell, wherein the chitosan dominateson the surface of the nanoparticles with positive surface charges. Inpractice, the Alzheimer's drug is encapsulated in the chitosan shellnanoparticle as described herein, wherein the nanoparticle is partiallycrosslinked (optionally) to enhance its biodurability. Then, thenanoparticles are intra-venously injected, whereby the nanoparticlespass to the brain in blood circulation. The chitosan shell of thenanoparticles adheres to the surface adjacent the tight junction in thebrain. Thereafter, the chitosan nanoparticle opens the tight junction,wherein the Alzheimer's drug is released for therapeutic treatment afterpassing the tight junction. In one embodiment, the nanoparticles are ina spherical shape having a mean particle size of about 50 to 250nanometers, preferably 150 nanometers to 250 nanometers.

Dalteparin is a low molecular weight heparin. It is marketed as Fragmin®by Pfizer Inc. Like other low molecular weight heparins, dalteparin isused for prophylaxis or treatment of deep vein thrombosis and pulmonaryembolism. The CLOT study, published in 2003, showed that in patientswith malignancy and acute venous thromboembolism, dalteparin was moreeffective than Coumadin in reducing the risk of recurrent embolicevents. Dalteparin is the only low molecular weight heparin shown to besafe in critically ill people with renal failure. Heparins are clearedby the kidneys, but studies have shown that dalteparin does notaccumulate even if kidney function is reduced.

In one example, intravenous administration of the nanoparticlescomprising chitosan shell substrate, polyanionic core substrate and atleast one bioactive agent for treating Alzheimer's disease in an animalsubject is typically performed with 10 mg to 40 mg of active agent perday over a period of one month to one year. The bioactive agent isselected from the group consisting of donepezile, rivastignine,galantamine, and/or those trade-named products, such as memantinehydrochloride (Axura® by Merz Pharmaceuticals), donepezil hydrochloride(Aricept® by Eisai Co. Ltd.), rivastigmine tartrate (Exelon® byNovartis), galantamine hydrochloride (Reminyl® by Johnson & Johnson),and tacrine hydrochloride (Cognex® by Parke Davis).

Some aspects of the invention relate to a nanoparticle with a coresubstrate comprising polyglutamic acids such as water soluble salt ofpolyglutamic acids (for example, ammonium salt) or metal salts ofpolyglutamic acid (for example, lithium salt, sodium salt, potassiumsalt, magnesium salt, and the like). In one embodiment, the polyglutamicacid may be selected from the group consisting of poly-α-glutamic acid,poly-L-α-glutamic acid, poly-γ-glutamic acid, poly-D-glutamic acid,poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid, poly-L-glutamic acid(manufactured by Sigma-Aldrich, St. Louis, Mo.), and PEG or PHEGderivatives of polyglutamic acid. Alginate is generallynon-biodegradable; however, it is stipulated that an alginate particlewith about 30-50 kDa molecular weight is kidney inert. Heparin withnegatively charged side-groups has a general chemical structure as shownbelow:

Some aspects of the invention relate to the negatively chargedglycosaminoglycans (GAGs) as the core substrate of the presentnanoparticles. GAGs may be complexed with a low-molecular-weightchitosan to form drug-carrier nanoparticles. GAGs may also conjugatewith the protein drugs as disclosed herein to enhance the bondingefficiency of the core substrate in the nanoparticles. Particularly, thenegatively charged core substrate (such as GAGs, heparin, PGA, alginate,and the like) of the nanoparticles of the present invention mayconjugate with chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF,MK, VEGF, KGF, bFGF, aFGF, MK, PTN, etc.

Chemical modification of an API involves the conjugation of the API (forexample, a peptide drug) via a chemical bond with specific functionalgroup(s) that often serve the twin purposes of stabilizing the APIagainst enzymatic degradation and enhancing its permeability across theintestinal epithelia. PEG modification (pegylation) is to improve theaqueous solubility and decrease the immunogenicity of an API. It hasbeen applied to prolong the half-life of peptide drugs administered byinjection. The increased circulation time was achieved through enhanceddrug stability in vivo, reduced renal clearance and prolonged localresidence at the injection site.

Another approach is to enable either chitosan or the negatively chargedsubstrate component of the instant nanoparticle formulation to conjugatewith moieties. In one embodiment, the amphiphilic moiety contained 1 ormore alkyl groups, together with 1 or more ethylene oxide (also calledethylene glycol) oligomers. Conjugation could occur via an amide bondand the carboxyl group present in the alkyl or ethylene oxide chains ofthe amphiphilic molecule.

Anti-Diabetic Drugs

The anti-diabetic drugs are broadly categorized herein asinsulin/insulin analogs and non-insulin anti-diabetic drugs. Thenon-insulin anti-diabetic drugs may include, but not limited to, insulinsensitizers, such as biguanides (for example, metformin, buformin,phenoformin, and the like), thiazolidinedione (TZDs; for example,pioglitazone, rivoglitazone, rosiglitazone, troglitazone, and the like),and dual PPAR agonists (for example aleglitazar, muraglitazar,tesaglitazar, and the like; PPAR is abbreviation for peroxisomeproliferator-activated receptor). The non-insulin anti-diabetic drugsmay also include, but not limited to, secretagogues, such assulfonylureas (for example, carbutamide, chlopropamide, gliclazide,tolbutamide, tolazamide, glipizide, glibenclamide, gliquidone,glyclopyramide, glimepiride, and the like), meglitinides (for example,nateglinide, repaglinide, mitiglinide, and the like), GLP-1 analogs (forexample, exenatide, liraglutide, albiglutide, taspoglutide, and thelike), and DPP-4 inhibitors (for example, alogliptin, linagliptin,saxagliptin, sitagliptin, vildagliptin, and the like; DPP-4 isabbreviation for inhibitor of dipeptidyl peptidase 4). Further, thenon-insulin anti-diabetic drugs may include, but not limited to,alpha-glucosidase inhibitors (for example, acarbose, miglitol,voglibose, and the like), amylin analog (for example, pramlintide andthe like), SGLT2 inhibitor (for example, dapagliflozin, remogliflozin,sergliflozin, and the like), benfluorex, and tolrestat. Here, Thesodium-glucose co-transporter type 2 (SGLT2) is a 672 amino acidhigh-capacity low-affinity transporter expressed in the S1 segment ofthe proximal tubule which is believed to mediate the majority of renalglucose reabsorption.

Clinical data have shown enhanced anti-diabetic efficiency (or glycemiccontrol) in an animal subject by co-administering two differentanti-diabetic drugs; for example, exenatide has been approved to beco-administered along with metformin, or a combination of metformin anda sulfonyurea, or thiazolidinediones (such as pioglitazone orrosiglitazone). In cholesterol management clinical trials, it wasreported that the combination of bezafibrate and diffunisol producedbetter clinical data than bezafibrate alone. Bezafibrate (marketed asBezalip and various other brand names) is a fibrate drug used for thetreatment of hyperlipidaemia. It helps to lower LDL cholesterol andtriglyceride in the blood, and increase HDL.

Some aspects of the invention relate to a therapeutic method of treatinga subject by co-administering at least two nanoparticle compositions,the first nanoparticle composition of the present invention comprising afirst bioactive agent, wherein the second nanoparticle composition ofthe present invention comprises a second bioactive agent that isdifferent from the first bioactive agent. In one example, the firstbioactive agent is exenatide in the first nanoparticles and the secondbioactive agent is metformin in the second nanoparticles. In oneembodiment, the delivery route of administering the first nanoparticlecomposition is different from the delivery route of administering thesecond of the at least two nanoparticle compositions. In one embodiment,both types of first and second nanoparticles are loaded in the samecapsules. In another embodiment, the first nanoparticles and the secondnanoparticles are loaded in separate different capsules. In a furtherembodiment, the first bioactive agent is compatible with the secondbioactive agent and optionally, the first bioactive agent enhances thetherapeutic effects of the second bioactive agent when they areco-administered to the subject.

Some aspects of the invention relate to a system of pharmaceuticalcomposition comprising two distinct types of bioactive nanoparticles,wherein the first type of bioactive nanoparticles comprises a shellportion that is dominated by positively charged chitosan, a core portionthat contains negatively charged substrate, wherein the negativelycharged substrate is at least partially neutralized with a portion ofthe positively charged chitosan and at least a first bioactive agent,and wherein the second type of bioactive nanoparticles comprises a shellportion that is dominated by positively charged chitosan, a core portionthat contains negatively charged substrate, wherein the negativelycharged substrate is at least partially neutralized with a portion ofthe positively charged chitosan and at least a second bioactive agent.In one embodiment, both types of the first and second nanoparticles areloaded in the same capsules for administering to a subject. In anotherembodiment, the first type of nanoparticles and the second type ofnanoparticles are loaded in separate different capsules in the systemfor co-administering to a subject.

Calceti et al. reported an in vivo evaluation of an oral insulin-PEGdelivery system (Eur J Pharma Sci 2004; 22:315-323). Insulin-PEG wasformulated into mucoadhesive tablets constituted by the thiolatedpolymer poly(acrylic acid)-cysteine. The therapeutic agent was releasedfrom these tablets within 5 hours in a sustained manner. In vivo, byoral administration to diabetic mice, the glucose levels were found todecrease significantly over the time. Further, Krauland et al. reportedanother oral insulin delivery study of thiolated chitosan-insulintablets on non-diabetic rats (J. Control. Release 2004, 95:547-555). Thedelivery tablets utilized 2-Iminothiolane covalently linked to chitosanto form chitosan-TBA (chitosan-4-thiobutylamidine) conjugate. After oraladministration of chitosan-TBA-insulin tablets to non-diabetic consciousrats, the blood glucose level decreased significantly for 24 hours;supporting the expected sustained insulin release of the presentlydisclosed nanoparticles herein through intestinal absorption. In afurther report by Morcol et al. (Int. J. Pharm. 2004; 277:91-97), anoral delivery system comprising calcium phosphate-PEG-insulin-caseinparticles displays a prolonged hypoglycemic effect after oraladministration to diabetic rats.

Pan et al. disclosed that chitosan nanoparticles improving theintestinal absorption of insulin in vivo (Int J Pharma 2002;249:139-147) with insulin-chitosan nanoparticles at a particle size of250-400 nm, a polydispersity index smaller than 0.1, positively chargedand stable. After administering the insulin-chitosan nanoparticles, itwas found that the hypoglycemic effect was prolonged with enhancedpharmacological bioavailability. Their data confirmed our observation asshown in FIGS. 11 and 12; however, the insulin loading capacity andinsulin association efficiency of the present invention aresubstantially higher for the chitosan-insulin nanoparticles with γ-PGAin the core as the core substrate.

Example No. 8 Insulin Nanoparticle Stability

FIG. 13 shows the stability of insulin-loaded nanoparticles of thepresent invention with an exemplary composition of CS 0.75 mg/ml, γ-PGA0.167 mg/ml, and insulin 0.083 mg/ml. The prepared insulin-loadednanoparticles suspended in deionized water are stable during storage upto 40 days. First (in FIG. 13), the insulin content in the nanoparticlestorage solution maintains at about a constant level of 9.5%. Thenanoparticle stability is further evidenced by the substantiallyconstant particle size at about 200 nm and substantially constant zetapotential of about +28 mV over the period of about 40 days. It iscontemplated that the insulin-containing nanoparticles of the presentinvention would further maintain their biostability when formulated in asoft gelcap or capsule configuration that further isolates thenanoparticles from environmental effects, such as sunlight, heat, airconditions, and the like. Some aspects of the invention provide a gelcappill or capsule containing a dosage of insulin nanoparticles effectiveamount of the insulin to treat or manage the diabetic animal subjects,wherein the stability of the insulin-containing nanoparticles is atleast 40 days, preferably more than 6 months, and most preferably morethan a couple of years.

By “effective amount of the insulin”, it is meant that a sufficientamount of insulin will be present in the dose to provide for a desiredtherapeutic, prophylatic, or other biological effect when thecompositions are administered to a host in single dosage forms. Thecapsule of the present invention may preferably comprise two-parttelescoping gelatin capsules. Basically, the capsules are made in twoparts by dipping metal rods in molten gelatin solution. The capsules aresupplied as closed units to the pharmaceutical manufacturer. Before use,the two halves are separated, the capsule is filled with powder (eitherby placing a compressed slug of powder into one half of the capsule, orby filling one half of the capsule with loose powder) and the other halfof the capsule is pressed on. The advantage of inserting a slug ofcompressed powder is that there is a superior control of weightvariation. The capsules may be enterically coated before filling thepowder or after securing both parts of the filled capsule together. Inone embodiment, the capsules further comprise a permeation enhancer,wherein the permeation enhancer is selected from the group consisting ofCa²⁺ chelators, bile salts, anionic surfactants, medium-chain fattyacids, phosphate esters, chitosan, and chitosan derivatives.

In another embodiment, the capsule may contain solubilizer, bubblingagent, emulsifier, or other pharmacopoeial excipients, such as GenerallyRecognized as Safe (GRAS). GRAS is a United States of America Food andDrug Administration (FDA) designation that a chemical or substance addedto food is considered safe by experts, and therefore exempted from theusual Federal Food, Drug, and Cosmetic Act (FFDCA) food additivetolerance requirements. The bubbling agent is the agent that emitscarbon dioxide gas when contacting liquid with a purpose to burst thecapsule or promote intimate contact of the capsule content with thesurrounding material outside of the capsule. For example, reaction ofsodium bicarbonate and an acid to give a salt and carbonic acid, whichreadily decomposes to carbon dioxide and water. The bubbling agent mayinclude sodium bicarbonate/citric acid mixture, Ac-Di-Sol, and the like.The chemical Ac-Di-Sol has the IUPAC name of acetic acid,2,3,4,5,6-pentahydroxyhexanal, sodium and a chemical formula ofC₈H₁₆NaO₈. An emulsifier is a substance that stabilizes an emulsion byincreasing its kinetic stability. One class of emulsifiers is known assurface active substances, or surfactants. Detergents are another classof surfactant emulsifier, and will physically interact with both oil andwater, thus stabilizing the interface between oil or water droplets insuspension. The most popular emulsions are non-ionic because they havelow toxicity. Cationic emulsions may also be used herein because theirantimicrobial properties.

Thus, for convenient and effective oral administration, pharmaceuticallyeffective amounts of the nanoparticles of this invention can betabletted with one or more excipient, encased in capsules such as gelcapsules, or suspended in a liquid solution and the like. Thenanoparticles can be suspended in a deionized solution or a similarsolution for parenteral administration. The nanoparticles may be formedinto a packed mass for ingestion by conventional techniques. Forinstance, the nanoparticles may be encapsulated as a “hard-filledcapsule” or a “soft-elastic capsule” using known encapsulatingprocedures and materials. The encapsulating material should be highlysoluble in gastric fluid so that the particles would be rapidlydispersed in the stomach after the capsule is ingested. Each unit dose,whether capsule or tablet, will preferably contain nanoparticles of asuitable size and quantity that provides pharmaceutically effectiveamounts of the nanoparticles. The applicable shapes and sizes ofcapsules may include round, oval, oblong, tube or suppository shape withsizes from 0.75 mm to 80 mm or larger. The volume of the capsules can befrom 0.05 cc to more than 5 cc. In one embodiment, the interior ofcapsules is treated to be hydrophobic or lipophilic.

Example No. 9 In Vitro Insulin Release Study

FIG. 14 show a representative protein drug (for example, insulin)release profile in a pH-adjusted solution for pH-sensitivity study withan exemplary composition of CS 0.75 mg/ml, γ-PGA 0.167 mg/ml, andinsulin 0.083 mg/ml in nanoparticles. In one embodiment, the exemplarycomposition may include each component at a concentration range of ±10%as follows: CS 0.75 mg/ml (a concentration range of 0.67 to 0.83 mg/ml),γ-PGA 0.167 mg/ml (a concentration range of 0.150 to 0.184 mg/ml), andinsulin 0.083 mg/ml (a concentration range of 0.075 to 0.091 mg/ml).First, solution of the insulin-loaded nanoparticles was adjusted to pH2.5 to simulate the gastric environment in a DISTEK-2230A container at37° C. and 100 rpm. Samples (n=5) were taken at a pre-determinedparticular time interval and the particle-free solution was obtained bycentrifuging at 22,000 rpm for 30 minutes to analyze the free orreleased insulin in solution by HPLC. Until the free insulin content inthe sample solution approaches about constant of 26% (shown in FIG. 14),the pH was adjusted to 6.6 to simulate the entrance portion of theintestine. The net released insulin during this particular time intervalis about (from 26% to 33%) 7%. In other words, the nanoparticles arequite stable (evidenced by minimal measurable insulin in solution) forboth the pH 2.5 and pH 6.6 regions.

To simulate the exit portion of the intestine, the insulin-containingnanoparticle solution was adjusted to pH 7.4. The remaining insulin(about 67%) was released from the nanoparticles at this time. Asdiscussed above, the insulin in nanoparticles would be more effective topenetrate the intestine wall in paracellular transport mode than thefree insulin because of the present invention of the nanoparticles withchitosan at the outer surface (preferential mucosal adhesion on theintestinal wall) and positive charge (enhancing paracellular tightjunction transport).

Since chitosan-shelled nanoparticles exhibit positive surface charge andpreferential mucoadhesive properties (both are required for enhancingparacellular permeation), some aspects of the invention relate to amethod of coating a nanoparticle (for example, a nanoparticle withlittle or no chitosan) with chitosan solution, resulting in achitosan-shelled nanoparticle with positive surface charge.

Example No. 10 In Vivo Study with Insulin-Loaded Fluorescence-LabeledNanoparticles

In the in vivo study, rats were injected with streptozotocin (STZ 75mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to inducediabetes. The blood from the rat's tail was analyzed with a commerciallyavailable glucometer for blood glucose. The blood glucose level onWistar male rats at no fasting (n=5) was measured at 107.2±8.1 mg/dL fornormal rats while the blood glucose level was at 469.7±34.2 mg/dL fordiabetic rats. In the animal study, diabetic rats were fasting for 12hours and subjected to four different conditions: (a) oral deionizedwater (DI) administration; (b) oral insulin administration at 30 U/kg;(c) oral insulin-loaded nanoparticles administration at 30 U/kg; and (d)subcutaneous (SC) insulin injection at 5 U/kg as positive control. Theblood glucose concentration from rat's tail was measured over the timein the study.

FIG. 15 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines (the base line was the glucose level in an animal subject withoutthe effect of insulin) for both oral DI administration and oral insulinadministration over a time interval of 8 hours appears relativelyconstant within the experimental measurement error range. It isillustrative that substantially all insulin from the oral administrationroute has been decomposed in rat stomach. As anticipated, the glucosedecrease for the SC insulin injection route appears in rat blood in thevery early time interval and starts to taper off after 3 hours in thisexemplary study.

The most important observation of the study comes from the oraladministration route with insulin-loaded nanoparticles. The bloodglucose begins to decrease from the base line at about 2 hours afteradministration and sustains a lower glucose level at more than 8 hoursinto study. It implies that the current insulin-loaded nanoparticles maymodulate the glucose level in animals in a sustained or prolongedeffective mode. Some aspects of the invention provide a method oftreating diabetes of an animal subject comprising orally administeringinsulin-containing nanoparticles with a dosage effective amount of theinsulin to treat the diabetes, wherein at least a portion of thenanoparticles comprises a positively charged shell substrate and anegatively charged core substrate. In one embodiment, the dosageeffective amount of the insulin to treat the diabetes comprises aninsulin amount of between about 15 units to 45 units per kilogram bodyweight of the animal subject, preferably 20 to 40 units, and mostpreferably at about 25 to 35 units insulin per kilogram body weight.

Some aspects of the invention relate to a novel nanoparticle system thatis composed of a low-MW CS and γ-PGA with CS dominated on the surfacesbeing configured to effectively open the tight junctions between Caco-2cell monolayers. The surface of the nanoparticles is characterized witha positive surface charge. In one embodiment, the nanoparticles of theinvention enables effective intestinal delivery for bioactive agent,including peptide, polypeptide, protein drugs, other large hydrophilicmolecules, and the like. Such polypeptide drugs can be any natural orsynthetic polypeptide that may be orally administered to a patient or ananimal subject.

Exemplary drugs include, but are not limited to, insulin; growthfactors, such as epidermal growth factor (EGF), insulin-like growthfactor (IGF), transforming growth factor (TGF), nerve growth factor(NGF), platelet-derived growth factor (PDGF), bone morphogenic protein(BMP), fibroblast growth factor and the like; hemophilia factors,somatostatin; somatotropin; somatropin; somatrem; calcitonin;parathyroid hormone; colony stimulating factors (CSF); clotting factors;tumor necrosis factors: interferons; interleukins; gastrointestinalpeptides, such as vasoactive intestinal peptide (VIP), cholecytokinin(CCK), gastrin, secretin, and the like; erythropoietins; growth hormoneand GRF; vasopressins; octreotide; pancreatic enzymes; dismutases suchas superoxide dismutase; thyrotropin releasing hormone (TRH); thyroidstimulating hormone; luteinizing hormone; LHRH; GHRH; tissue plasminogenactivators; macrophage activator; chorionic gonadotropin; heparin;atrial natriuretic peptide; hemoglobin; retroviral vectors; relaxin;cyclosporin; oxytocin; vaccines; monoclonal antibodies; and the like;and analogs and derivatives of these compounds.

Triptorelin (acetate or pamoate), a decapeptide(pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), is agonadotropin-releasing hormone agonist (GnRH agonist). By causingconstant stimulation of the pituitary, it decreases pituitary secretionof gonadotropins luteinizing hormone (LH) and follicle stimulatinghormone (FSH). Like other GnRH agonists, triptorelin may be used in thetreatment of hormone-responsive cancers such as prostate cancer orbreast cancer, precocious puberty, estrogen-dependent conditions (suchas endometriosis or uterine fibroids), and in assisted reproduction.Triptorelin is marketed under the brand names Decapeptyl (Ipsen) andDiphereline and Gonapeptyl (Ferring Pharmaceuticals). In the UnitedStates, it is sold by Pfizer as Trelstar. Its systematic (IUPAC) name is5-oxo-D-prolyl-L-histidyl-Ltryptoph-L-seryl-Ltyrosyl-3-(1H-indol-2-yl)-L-alanylleucyl-L-arginyl-L-prolylglycinamide. It has a chemical formulaC₆₄H₈₂N₁₈O₁₃ with a molecular mass 1311.5 g/mol.

Gemtuzumab ozogamicin (marketed by Wyeth as Mylotarg®) is a monoclonalantibody used to treat acute myelogenous leukemia. It is a monoclonalantibody to CD33 linked to a cytotoxic agent, calicheamicin. CD33 isexpressed in most leukemic blast cells but also in normal hematopoieticcells, the intensity diminishing with maturation of stem cells. Thegemtuzumab ozogamicin has been evaluated in the nanoparticle formulationof the present invention, showing typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge, and a narrow polydispersity index.

The bioactive agent of the present invention may also be selected fromgroup consisting of oxytocin, vasopressin, adrenocorticotrophic hormone,prolactin, luliberin or luteinising hormone releasing hormone, growthhormone, growth hormone releasing factor, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, gramicidines, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines. Growthhormone (GH) is a peptide hormone that stimulates growth and cellreproduction in humans and other animals. It is a 191-amino acid, singlechain polypeptide hormone which is synthesized, stored, and secreted bythe somatotroph cells within the lateral wings of the anterior pituitarygland. Somatotrophin refers to the growth hormone produced natively inanimals, the term somatropin refers to growth hormone produced byrecombinant DNA technology,^([1]) and is abbreviated “rhGH” in humans.

In another embodiment, the nanoparticles of the invention increase theabsorption of bioactive agents across the blood brain barrier and/or thegastrointestinal barrier. In still another embodiment, the nanoparticleswith chitosan dominant at an outer layer that show positive surfacecharge serve as an enhancer in enhancing drug (bioactive agent)permeation of an administered bioactive agent when the bioactive agentand nanoparticles are orally administrated in a two-component system, ororally administered substantially simultaneously.

Example No. 11 Epithelial Permeation and Absorption Enhancers

Chitosan and its derivatives may function as epithelial absorptionenhancers. Chitosan, when protonated at an acidic pH, is able toincrease the permeability of peptide drugs across mucosal epithelia.Some aspects of the invention provide co-administration of nanoparticlesof the present invention and at least one permeation enhancer (innon-nanoparticle form or nanoparticle form). In one embodiment, thenanoparticles can be formulated by co-encapsulation of at least onepermeation enhancer and at least one bioactive agent, with an option ofadding other components. In one embodiment, the nanoparticles furthercomprise a permeation enhancer. The permeation enhancer may be selectedfrom the group consisting of Ca²⁺ chelators, bile salts (for example,sodium cholate, sodium taurocholate, sodium taurodeoxycholate, sodiumtaurodihydrofusidate, sodium chenodeoxycholate, sodium glycocholate,sodium deoxycholate, sodium lithocholate, and the like), surfactants,medium-chain fatty acids, phosphate esters, and chitosan or chitosanderivatives. In one embodiment, the surfactant may include non-ionicsurfactant (for example, fatty alcohols, polyoxypropylene glycol alkylethers, glycerol alkyl esters, and the like), anionic surfactant (forexample, alkyl sulfate, alkyl benzene sulfonate, alkyl ether phosphate,alkyl carboxylates, and the like), cationic surfactant (for example,pH-dependent primary, secondary or tertiary amine, permanently chargedquaternary ammonium cations, and the like), or zwitterionic surfactant.

In some embodiments, the nanoparticles of the present invention, or withat least one permeation enhancer are loaded in a soft gel, pill, tablet,chewable, or capsule, or loaded in the enteric coated counterpart of thesoft gel, pill, tablet, chewable, or capsule. The enhancers and thenanoparticles would arrive at the tight junction about the same time toenhance transiently opening the tight junction. In another embodiment,the at least one permeation enhancer is co-enclosed within the shell ofthe nanoparticles of the present invention. Therefore, some brokennanoparticles or fragments would release enhancers to assist thenanoparticles to open the tight junctions of the epithelial layers. Inan alternate embodiment, the at least one enhancer is enclosed within asecond nanoparticle having positive surface charges, particularly achitosan-type nanoparticle, wherein the second nanoparticle isformulated without any bioactive agent or with a different bioactiveagent from that bioactive agent in the first nanoparticle. When thedrug-containing first nanoparticles of the present invention areco-administered with the above-identified second nanoparticles orally,the enhancers within the second nanoparticles are released in thegastrointestinal tract to assist the drug-containing first nanoparticlesto open and pass the tight junction or facilitate enhanced drugabsorption and transport.

Example No. 12 Nanoparticles Loaded with Exenatide

Exenatide is a member of the class of drugs known as incretin mimetics.Exenatide and pramlintide belong to a group of non-insulin injectablesfor treatment of diabetes. Exenatide has a molecular formula ofC₁₈₄H₂₈₂N₅₀O₆₀S with a molecular mass of about 4186.6 g/mol and an CASno. 141732-76-5. Exenatide is suitable to be incorporated in the coreportion of chitosan-shelled nanoparticles, wherein the core portion mayinclude positively charged chitosan and negatively charged coresubstrate, such as γ-PGA or α-PGA, with the option of additional TPP andMgSO₄ in the core portion. In preparation, nanoparticles were obtainedupon addition of a mixture of γ-PGA plus exenatide aqueous solution (pH7.4, 2 ml), using a pipette during the addition stage (0.5-5 ml,PLASTIBRAND®, BrandTech Scientific Inc., Germany), into a low-MW CSaqueous solution (pH 6.0, 10 ml) at concentrations higher than 0.10% byw/v under magnetic stirring at room temperature to ensure positivesurface charge. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Exenatide is wholly or substantially totallyencapsulated within the nanoparticles. Supernatants were discarded andnanoparticles were resuspended in deionized water as the solutionproducts. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge and a narrow polydispersity index. In oneembodiment, it may further be encapsulated in capsules. In oneembodiment, the interior surface of the capsule is treated to belipophilic or hydrophobic. In another embodiment, the exterior surfaceof the capsule is enteric-coated. In a preferred embodiment, thenanoparticles are further freeze-dried, optionally being mixed withtrehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.

Glucagon-like peptide-1 (GLP-1) is derived from the transcriptionproduct of the proglucagon gene. The major source of GLP-1 in the bodyis the intestinal L cell that secretes GLP-1 as a gut hormone. Thebiologically active forms of GLP-1 are GLP-1-(7-37) and GLP-1-(7-36)NH2.GLP-1 secretion by L cells is dependent on the presence of nutrients inthe lumen of the small intestine. The secretagogues (agents that causeor stimulate secretion) of this hormone include major nutrients likecarbohydrate, protein and lipid. Once in the circulation, GLP-1 has ahalf-life of less than 2 minutes, due to rapid degradation by the enzymedipeptidyl peptidase-4 (DPP-4). Commercial GLP-1 ELISA kits aregenerally available for GLP-1 assay.

Exenatide (marketed as Byetta) is the first of a new class ofmedications (incretin mimetics) approved for the treatment of type 2diabetes. It is manufactured and marketed by Amylin Pharmaceuticals andEli Lilly and Company. Exenatide is a synthetic version of exendin-4, ahormone in the saliva of the Gila monster, a lizard native to severalSouthwestern American states. It displays properties similar to humanGLP-1. Exenatide is a 39-amino-acid peptide that mimics the GLP-1incretin, an insulin secretagogue with glucoregulatory effects. While itmay lower blood glucose levels on its own, it can also be combined withother medications such as pioglitazone, metformin, sulfonylureas, and/orinsulin (not FDA approved yet) to improve glucose control. The approveduse of exenatide is with sulfonylureas, metformin or thiazolinediones.The medication is injected subcutaneously twice per day using apre-filled pen device.

Typical human responses to exenatide include improvements in the initialrapid release of endogenous insulin, suppression of pancreatic glucagonrelease, delayed gastric emptying, and reduced appetite—all of whichfunction to lower blood glucose. Whereas some other classes of diabetesdrugs such as sulfonylureas, thiazolinediones, and insulin are oftenassociated with weight gain, Byetta often is associated with significantweight loss. Unlike sulfonylureas and meglitinides, exenatide onlyincreases insulin synthesis and secretion in the presence of glucose,lessening the risk of hypoglycemia. Byetta is also being used by somephysicians to treat insulin resistance.

Example No. 13 Nanoparticles Loaded with Pramlintide

Pramlintide is a synthetic amylin analogue (marketed as Symlin). Amylinis a natural, pancreatic islet peptide that is normally secreted withinsulin in response to meals. It has several beneficial effects onglucose homeostasis: suppression of glucagon secretion, delaying ofgastric emptying, and promotion of satiety. It is currently given beforemeals, in a separate subcutaneous injection but usually in conjunctionwith insulin. Pramlintide has a molecular formula of C₁₇₁H₂₆₉N₅₁O₅₃S₂with a molecular mass of about 3951.4 g/mol and an CAS no. 151126-32-8.Pramlintide (positively charged) is currently delivered as an acetatesalt. Pramlintide is suitable to be incorporated in a core portion of achitosan-shelled nanoparticles, wherein the core portion may includepositively charged chitosan and negatively charged core substrate, suchas γ-PGA or α-PGA, with an option for additional TPP and MgSO₄ in thecore portion. In other words, pramlintide may replace at least a portionof positively charged chitosan in the core by interacting with thenegatively core substrate, such as PGA, heparin or the like. Inpreparation, nanoparticles were obtained upon the addition of a mixtureof γ-PGA plus pramlintide aqueous solution (pH 7.4, 2 ml), using apipette during the addition step (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at concentrations higher than 0.10% by w/v under magnetic stirringat room temperature to ensure positive surface charge.

Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1hour. Pramlintide is wholly or substantially totally encapsulated withinthe nanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products. Thenanoparticles thus obtained via the simple and mild ionic-gelationmethod described herein show typical characteristics in a spheroidalconfiguration with a particle size of between about 50 to 400 nm, apositive surface charge and a narrow polydispersity index. In oneembodiment, it may further be encapsulated in capsules. In oneembodiment, the interior surface of the capsule is treated to belipophilic or hydrophobic. In another embodiment, the exterior surfaceof the capsule is enteric-coated. In a preferred embodiment, thenanoparticles are further freeze-dried, and can be optionally mixed withtrehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.

Pramlintide is an analogue of amylin, a small peptide hormone that isreleased into the bloodstream after a meal by the β-cells of thepancreas along with insulin. Like insulin, amylin is deficient inindividuals with diabetes. By augmenting endogenous amylin, pramlintideaids in the absorption of glucose by slowing gastric emptying, promotingsatiety via hypothalamic receptors (different receptors than GLP-1), andinhibiting inappropriate secretion of glucagon, a catabolic hormone thatopposes the effects of insulin and amylin.

Example No. 14 Nanoparticles Loaded with Complexed Calcitonin

Calcitonin is a protein drug that therapeutically serves as calciumregulators for treating osteoporosis (J. Pharm. Pharmacol. 1994;46:547-552). Calcitonin has a molecular formula of C₁₄₅H₂₄₀N₄₄O₄₈S₂ witha molecular weight of about 3431.9 and an isoelectric point of 8.7. Thenet charge for calcitonin at pH 17.4 is positive that makes it suitablefor complexing or conjugating with negatively charged core substrate,such as γ-PGA or α-PGA. In preparation, nanoparticles were obtained uponthe addition of a mixture of γ-PGA and calcitonin aqueous solution (pH7.4, 2 ml), using a pipette during the addition step (0.5-5 ml,PLASTIBRAND®, BrandTech Scientific Inc., Germany), into a low-MW CSaqueous solution (pH 6.0, 10 ml) at concentrations higher than 0.10% byw/v under magnetic stirring at room temperature to ensure positivesurface charge. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Calcitonin is wholly or substantially totallyencapsulated within the nanoparticles. Supernatants were discarded andnanoparticles were resuspended in deionized water as the solutionproducts, further encapsulated in capsules or further treated with anenteric coating. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge and a narrow polydispersity index.

Example No. 15 Nanoparticles Loaded with Teriparatide

Teriparatide (Forteo®) is a recombinant form of parathyroid hormone,used in the treatment of some forms of osteoporosis. It is manufacturedand marketed by Eli Lilly and Company. Currently teriparatide isadministered by injection once a day in the thigh or abdomen. Therecommended dose is 20 μg per day. Teriparatide has the chemical formulaC₁₈₁H₂₉₁N₅₅O₅₁S₂ with a molecular mass of 4117.72 g/mol. Teriparatide isthe portion of human parathyroid hormone (PTH), amino acid sequence 1through 34 of the complete molecule which contains amino acid sequence 1to 84. Endogenous PTH is the primary regulator of calcium and phosphatemetabolism in bone and kidneys. Daily injections of teriparatidestimulate new bone formation leading to increased bone mineral density.

Teriparatide is the first FDA approved agent for the treatment ofosteoporosis that stimulates new bone formation. In one exemplarypreparation, nanoparticles were obtained upon addition of a mixture ofγ-PGA plus teriparatide aqueous solution (2 ml), using a pipette (0.5-5ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany) during theaddition step, into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Teriparatideis wholly or substantially totally encapsulated within thenanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products, furtherencapsulated in capsules/enteric capsules or further treated withlyophilization. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge and a narrow polydispersity index.

Example No. 16 Nanoparticles Loaded with Vancomycin

Vancomycin is a protein drug that serves therapeutically as anantibiotic against bacterial pathogens. Vancomycin has a molecularformula of C₆₆H₇₅N₉O₂₄ with a molecular weight of about 1485.7 and anisoelectric point of 5.0. The net charge for vancomycin at pH 7.4 isnegative, which is suitable to complex or conjugate with a portion ofnegatively charged shell substrate, such as chitosan. In preparation,nanoparticles were obtained upon addition of a mixture of γ-PGA andvancomycin aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,PLASTIBRAND®, BrandTech Scientific Inc., Germany), into a low-MW CSaqueous solution (pH 6.0, 10 ml) with excess concentrations undermagnetic stirring at room temperature, wherein CS concentration isprovided sufficiently to conjugate vancomycin, to counterbalance γ-PGA,and exhibit positive surface charge for the nanoparticles. Nanoparticleswere collected by ultracentrifugation at 38,000 rpm for 1 hour.Vancomycin is wholly or substantially totally encapsulated within thenanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products, furtherencapsulated in capsules or further treated with an enteric coating oncapsules. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge and a narrow polydispersity index.

Tigecycline is an glycylcycline antibiotic developed and marketed byWyeth under the brand name Tygacil®. It was developed in response to thegrowing prevalence of antibiotic resistance in bacteria such asStaphylococcus aureus. It has a systematic (IUPAC) nameN-[(5aR,6aS,7S,9Z,10aS)-9-[amino(hydroxy)methylidene]-4,7-bis(dimethylamino)-1,10a,12-trihydroxy-8,10,11-trioxo-5,5a,6,6a,7,8,9,10,10a,11-decahydrotetracen-2-yl]-2-(tert-butylamino)acetamide.The chemical formula is C₂₉H₃₉N₅O₈ with a molecular mass 585.65 g/mol.The drug inhibits the bacterial 30S ribosome and is bacteriostatic.Tigecycline is active against many Gram-positive bacteria, Gram-negativebacteria and anaerobes—including activity against methicillin-resistantStaphylococcus aureus (MRSA) and multi-drug resistant strains ofAcinetobacter baumannii. Tigecycline is currently given by slowintravenous infusion (30 to 60 minutes). A single dose of 100 mg isgiven first, followed by 50 mg every twelve hours after that.

Lincomycin is a lincosamide antibiotic that comes from the actinomycesStreptomyces lincolnensis. It has been structurally modified by thionylchloride to its more commonly known 7-chloro-7-deoxy derivative,clindamycin. Its systematic (IUPAC) name is(2S,4R)—N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(methylsulfanyl)oxan-2-yl]propyl]-1-methyl-4-propylpyrrolidine-2-carboxamide.Its chemical formula is C₁₈H₃₄N₂O₆S with a molecular mass 406.538 g/mol.

An antibiotic is a substance or compound (also called chemotherapeuticagent) that kills or inhibits the growth of bacteria. Antibiotics belongto the group of antimicrobial compounds, i.e., those for treatinginfections caused by microorganisms, including viruses, fungi, andprotozoa. Penicillin is a group of antibiotics derived from Penicilliumfungi. They are Beta-lactam antibiotics used in the treatment ofbacterial infections caused by susceptible, usually Gram-positive,organisms. The term Penam is used to describe the core skeleton of amember of a penicillin antibiotic. This skeleton has the molecularformula R—C₉H₁₁N₂O₄S, where R is a variable side chain. Ampicillin is abeta-lactam antibiotic that has been used extensively to treat bacterialinfections since 1961. It is considered part of the aminopenicillinfamily and is roughly equivalent to amoxicillin in terms of spectrum andlevel of activity. It can sometimes result in non-allergic reactionsthat range in severity from a rash (e.g. patients with mononucleosis) topotentially lethal anaphylaxis. Ampicillin is closely related toamoxicillin, another type of penicillin, and both are used to treaturinary tract infections, otitis media, uncomplicated community-acquiredpneumonia, Haemophilus influenzae, salmonellosis and Listeriameningitis. Other types include Penicillin V, Procaine benzylpenicillin,and Benzathine benzylpenicillin.

Some aspects of the invention relate to a method of treating infectionscaused by microorganisms in an animal subject, the method comprisingadministering nanoparticles composed of an antibiotic, chitosan, and acore portion of negatively charged substrate, wherein a surface of thenanoparticles is dominated by the chitosan. In one embodiment, themethod of administering the nanoparticles may comprise an oral route,intravenous route, subcutaneous injection route, intramuscular route,buccal route, intranasal route, parenteral route, and the like.

Piperacillin is an extended spectrum beta-lactam antibiotic of theureidopenicillin class. It is normally used together with abeta-lactamase inhibitor such as tazobactam, which is commerciallyavailable. The combination has activity against many Gram-positive andGram-negative pathogens and anaerobes, including Pseudomonas aeruginosa.The systematic (IUPAC) name is(2S,5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]-2-phenyl-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid. Its chemical formula is C₂₃H₂₆N₅O₇S with a molecular mass 516,548g/mol. Piperacillin is not absorbed orally, and must therefore be givenby intravenous or intramuscular injection; piperacillin/tazobactam isadministered intravenously every 6 or 8 hours; the drug may also begiven by continuous infusion, but this has not been shown to besuperior.

An aminoglycoside is a molecule composed of a sugar group and an aminogroup. Several aminoglycosides function as antibiotics that areeffective against certain types of bacteria. They include amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin, and apramycin.

Aminoglycosides have several potential antibiotic mechanisms, some asprotein synthesis inhibitors, although their exact mechanism of actionis not fully known: (1) They interfere with the proofreading process,causing increased rate of error in synthesis with premature termination;(2) Also, there is evidence of inhibition of ribosomal translocationwhere the peptidyl-tRNA moves from the A-site to the P-site; (3) Theycan also disrupt the integrity of bacterial cell membrane.

Aminoglycosides bind to the bacterial 30S ribosomal subunit; some workby binding to the SOS subunit. There is a significant relationshipbetween the dose administered and the resultant plasma level in blood.Therapeutic drug monitoring (TDM) is necessary to obtain the correctdose. These agents exhibit a post-antibiotic effect in which there is noor very little drug level detectable in blood, but there still seems tobe inhibition of bacterial re-growth. This is due to strong,irreversible binding to the ribosome, and remains intracellular longafter plasma levels drop. This allows a prolonged dosage interval.Depending on their concentration, they act as bacteriostatic orbactericidal agents.

The protein synthesis inhibition of aminoglycosides does not usuallyproduce a bactericidal effect, let alone a rapid one as is frequentlyobserved on susceptible Gram-negative bacilli. Aminoglycosidescompetitively displace cell biofilm-associated Mg²⁺ and Ca²⁺ that linkthe polysaccharides of adjacent lipopolysaccharide molecules. The resultis shedding of cell membrane blebs, with formation of transient holes inthe cell wall and disruption of the normal permeability of the cellwall. This action alone may be sufficient to kill most susceptibleGram-negative bacteria before the aminoglycoside has a chance to reachthe 30S ribosome.

Traditionally, the antibacterial properties of aminoglycosides werebelieved to result from inhibition of bacterial protein synthesisthrough irreversible binding to the 30S bacterial ribosome. Thisexplanation, however, does not account for the potent bactericidalproperties of these agents, since other antibiotics that inhibit thesynthesis of proteins (such as tetracycline) are not bactericidal.Recent experimental studies show that the initial site of action is theouter bacterial membrane. The cationic antibiotic molecules createfissures in the outer cell membrane, resulting in leakage ofintracellular contents and enhanced antibiotic uptake. This rapid actionat the outer membrane probably accounts for most of the bactericidalactivity.

Energy is needed for aminoglycoside uptake into the bacterial cell.Anaerobes have less energy available for this uptake, so aminoglycosidesare less active against anaerobes. Aminoglycosides are useful primarilyin infections involving aerobic, gram-negative bacteria, such asPseudomonas, Acinetobacter, and Enterobacter. In addition, someMycobacteria, including the bacteria that cause tuberculosis, aresusceptible to aminoglycosides. The most frequent use of aminoglycosidesis empiric therapy for serious infections such as septicemia,complicated intraabdominal infections, complicated urinary tractinfections, and nosocomial respiratory tract infections. Usually, oncecultures of the causal organism are grown and their susceptibilitiestested, aminoglycosides are discontinued in favor of less toxicantibiotics.

Streptomycin was the first effective drug in the treatment oftuberculosis, though the role of aminoglycosides such as streptomycinand amikacin has been eclipsed (because of their toxicity andinconvenient route of administration) except for multiple drug resistantstrains.

Infections caused by gram-positive bacteria can also be treated withaminoglycosides, but other types of antibiotics are more potent and lessdamaging to the host. In the past, the aminoglycosides have been used inconjunction with beta-lactam antibiotics in streptococcal infections fortheir synergistic effects, particularly in endocarditis. One of the mostfrequent combinations is ampicillin (a beta-lactam, orpenicillin-related antibiotic) and gentamicin. Often, hospital staffrefer to this combination as “amp and gent” or more recently called “penand gent” for penicillin and gentamicin. Aminoglycosides are mostlyineffective against anaerobic bacteria, fungi and viruses.

Experimentation with aminoglycosides as a treatment of cystic fibrosis(CF) has shown some promising results. CF is caused by a mutation in thegene coding for the cystic fibrosis transmembrane conductance regulator(CFTR) protein. In approximately 10% of CF cases the mutation in thisgene causes its early termination during translation, leading to theformation of is truncated and non-functional CFTR protein. It isbelieved that gentamicin distorts the structure of the ribosome-RNAcomplex, leading to a misreading of the termination cordon, causing theribosome to “skip” over the stop sequence and to continue with thenormal elongation and production of the CFTR protein. The treatment isstill experimental but showed improvement in cells from CF patients withsusceptible mutations.

Since they are not absorbed from the gut via conventional transcellularpermeability, they are administered intravenously and intramuscularly.Some are used in topical preparations for wounds. Oral administrationcan be used for gut decontamination (e.g. in hepatic encephalopathy).Tobramycin may be administered in a nebulized form. Some aspects of theinvention relate to a nanoparticle formulation as disclosed herein toenhance absorption via paracellular permeation route.

Temsirolimus is an intravenous drug for the treatment of renal cellcarcinoma (RCC), developed by Wyeth Pharmaceuticals and approved by theFDA in late May 2007. It is a derivative of sirolimus and is sold asTorisel. Its chemical formula is C₅₆H₈₇NO₁₆ with a molecular mass1030.28 g/mol. mTOR (mammalian target of rapamycin) is a kinase enzymeinside the cell that collects and interprets the numerous and variedgrowth and survival signals received by tumor cells. When the kinaseactivity of mTOR is activated, its downstream effectors, the synthesisof cell cycle proteins such as cyclin D and hypoxia-inducible factor-1a(HIF-1a) are increased. HIF-1a then stimulates VEGF. Temsirolimus is aspecific inhibitor of mTOR and interferes with the synthesis of proteinsthat regulate proliferation, growth, and survival of tumor cells.Treatment with temsirolimus leads to cell cycle arrest in the G1 phase,and also inhibits tumor angiogenesis by reducing synthesis of VEGF. Therecommended dose of temsirolimus is 25 mg IV infused over 30-60 minutesonce per week.

Some aspects of the invention relate to a method of enhancing epithelialpermeation of bioactive agents configured and adapted for delivering atleast one bioactive agent in an animal subject comprising administeringnanoparticles composed of γ-PGA and chitosan, wherein the nanoparticlesare loaded with a therapeutically effective amount or dose of the atleast one bioactive agent. The nanoparticle of the present invention isan effective intestinal delivery system for peptide and protein drugsand other large hydrophilic molecules. In a further embodiment, thebioactive agent is selected from the group consisting of proteins,peptides, nucleosides, nucleotides, antiviral agents, antineoplasticagents, antibiotics, antiepileptic drug, and anti-inflammatory drugs. Ina further embodiment, the bioactive agent is selected from the groupconsisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine,enkephalin, tyrotropin releasing hormone (TRH), follicle stimulatinghormone (FSH), luteinizing hormone (LH), vasopressin and vasopressinanalogs, catalase, superoxide dismutase, interleukin-II (IL2),interleukin-11 (IL-11), interferon, colony stimulating factor (CSF),tumor necrosis factor (TNF) and melanocyte-stimulating hormone.

In a further embodiment, the bioactive agent is an Alzheimer antagonist.In one embodiment, the antiepileptic drug may include Neurontin(gabapentin), Lamictal (lamotrigine), Febatol (felbamate), Topamax(topiramate), Cerebyx (fosphenyloin), Dilantin (phenyloin), Depakene(valproic acid), Tegretol (carbamazepine), carbamazepine epoxide, Vimpat(lacosamide) and phenobarbitol. Fosphenyloin (Cerebyx by Parke-Davis;Prodilantin by Pfizer Holding France) is a water-soluble phenyloinprodrug used only in hospitals for the treatment of epileptic seizuresthrough parental delivery. Fosphenyloin has systematic (IUPAC) name of(2,5-dioxo-4,4-diphenyl-imidazolidin-1-yl) methoxyphosphonic acid. Ithas the chemical formula of C₁₆H₁₅N₂O₆P with molecular mass 362.274g/mol.

Example No. 17 Nanoparticles Loaded with Heparin

Heparin is a negatively charged drug that serves therapeutically as ananti-coagulant. Heparin is generally administered by intravenousinjection. Some aspects of the invention relate to heparin nanoparticlesfor oral administration or subcutaneous administration. In a furtherembodiment, heparin serves as at least a portion of the core substratewith chitosan as shell substrate, wherein heparin conjugates with atleast one bioactive agent as disclosed herein. In preparation,nanoparticles were obtained upon addition of heparin Leo aqueoussolution (2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) with excess concentrations under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Heparin is wholly or substantially totallyencapsulated within the nanoparticles. The nanoparticles thus obtainedvia the simple and mild ionic-gelation method described herein showtypical characteristics in a spheroidal configuration with a particlesize of between about 50 to 400 nm, a positive surface charge and anarrow polydispersity index. Table 4 shows the conditions of solutionpreparation and the average nanoparticle size.

TABLE 4 Heparin conc. @ Chitosan conc. @ Particle size Conditions 2 ml10 ml (nm) A 200 iu/ml 0.09% 298.2 ± 9.3 B 100 iu/ml 0.09% 229.1 ± 4.5 C 50 iu/ml 0.09% 168.6 ± 1.7 D  25 iu/ml 0.09% 140.1 ± 2.3

To evaluate the pH stability of the heparin-containing nanoparticlesfrom Example no. 17, the nanoparticles from Condition D in Table 4 aresubjected to various pH level for 2 hours (sample size=7). Table 5 showsthe average size, size distribution (polydispersity index: PI) and zetapotential (Zeta) of the nanoparticles at the end of 2 hours undervarious pH environments. The data shows the nanoparticles are relativelystable. In one embodiment, the nanoparticles of the present inventionmay include heparin, heparin sulfate, small molecular weight heparin,and heparin derivatives.

TABLE 5 pH Deionized 1.5 2.6 6.6 7.4 water @5.9 Size 150 ± 9  160 ± 12 153 ± 2  154 ± 4  147 ± 5  (nm) PI 0.54 ± 0.03 0.50 ± 0.04 0.08 ± 0.020.32 ± 0.03 0.37 ± 0.02 Zeta (+) 15 ± 2  33 ± 6   15 ± 0.1  11 ± 0.2 18± 4 

In a further embodiment, a pharmaceutically effective amount of growthfactor such as bFGF is added to heparin Leo aqueous solution before thepipetting step in Example No. 15. In our laboratory, growth factors andproteins with pharmaceutically effective amounts have been successfullyconjugated with heparin to form nanoparticles of the present inventionwith chitosan as the shell substrate, wherein the growth factor isselected from the group consisting of Vascular Endothelial Growth Factor(VEGF), Vascular Endothelial Growth Factor 2 (VEGF2), basic FibroblastGrowth Factor (bFGF), Vascular Endothelial Growth Factor 121 (VEGF121),Vascular Endothelial Growth Factor 165 (VEGF165), Vascular EndothelialGrowth Factor 189 (VEGF189), Vascular Endothelial Growth Factor 206(VEGF206), Platelet Derived Growth Factor (PDGF), Platelet DerivedAngiogenesis Factor (PDAF), Transforming Growth Factor-13 (TGF-13),Transforming Growth Factor-α (TGF-α), Platelet Derived Epidermal GrowthFactor (PDEGF), Platelet Derived Wound Healing Formula (PDWHF),epidermal growth factor, insulin-like growth factor, acidic FibroblastGrowth Factor (aFGF), human growth factor, and combinations thereof; andthe protein is selected from the group consisting of haemagglutinin(HBHA), Pleiotrophin, buffalo seminal plasma proteins, and combinationsthereof.

In a co-pending application, U.S. patent application Ser. No. 10/916,170filed Aug. 11, 2004, it is disclosed that a biomaterial with free aminogroups of lysine, hydroxylysine, or arginine residues within biologictissues is crosslinkable with genipin, a crosslinker (Biomaterials 1999;20:1759-72). It is also disclosed that the crosslinkable biomaterial maybe crosslinked with a crosslinking agent or with light, such asultraviolet irradiation, wherein the crosslinkable biomaterial may beselected from the group consisting of collagen, gelatin, elastin,chitosan, NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biologicalsealant, and the like. Further, it is disclosed that a crosslinkingagent may be selected from the group consisting of genipin, itsderivatives, analog (for example, aglycon geniposidic acid),stereoisomers and mixtures thereof. In one embodiment, the crosslinkingagent may further be selected from the group consisting of epoxycompounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethylsuberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide,reuterin, ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers, and the like.

In one embodiment, it is disclosed that loading a drug onto achitosan-containing biological material crosslinked with genipin orother crosslinking agent may be used as biocompatible drug carriers fordrug slow-release or sustained release. Several biocompatible plasticpolymers or synthetic polymers have one or more amine group in theirchemical structures, for example poly(amides) or poly(ester amides). Theamine group may become reactive toward a crosslinking agent, such asglutaraldehyde, genipin or epoxy compounds of the present invention. Inone embodiment, the nanoparticles comprised of crosslinkable biomaterialis crosslinked, for example up to about 50% degree or more ofcrosslinking, preferably about 1 to about 20% degree of crosslinking ofthe crosslinkable components of the biomaterial, enabling sustainedbiodegradation of the biomaterial and/or sustained drug release.

By modifying the chitosan structure to alter its charge characteristics,such as grafting the chitosan with EDTA, methyl, N-trimethyl, alkyl (forexample, ethyl, propyl, butyl, isobutyl, etc.), polyethylene glycol(PEG), or heparin (including low molecular weight heparin, regularmolecular weight heparin, and genetically modified heparin), the surfacecharge density (zeta potential) of the CS-7 PGA nanoparticles may becomemore pH resistant or hydrophilic. In one embodiment, the chitosan isgrafted with polyacrylic acid. In one embodiment, the chitosan employedis N-trimethyl chitosan (TMC), low MW-chitosan, EDTA-chitosan, chitosanderivatives, and/or combinations thereof. An exemplary chemicalstructure for EDTA-chitosan is shown below:

By way of illustration, trimethyl chitosan chloride might be used informulating the CS-γ PGA nanoparticles for maintaining its sphericalbiostability at a pH lower than 2.5, preferably at a pH as low as 1.0.Some aspects of the invention provide a drug-loaded chitosan-containingbiological material crosslinked with genipin or other crosslinking agentas a biocompatible drug carrier for enhancing biostability at a pH lowerthan 2.5, preferably within at a pH as low as 1.0.

It is known that the pKa values of CS (amine groups) and γ-PGA(carboxylic groups) are 6.5 and 2.9, respectively. NPs were prepared inDI water (pH 6.0). At pH 6.0, CS (TMC25) and γ-PGA were ionized. Theionized CS (TMC25) and γ-PGA could form polyelectrolyte complexes, whichresulted in a matrix structure with a spherical shape. At pH 1.2-2.0,most carboxylic groups on γ-PGA were in the form of —COOH. Hence, therewas little electrostatic interaction between CS (TMC25) and γ-PGA; thusNPs became disintegrated (Table 6). Similarly, at pH values above 6.6,the free amine groups on CS (TMC25) were deprotonated; thus leading tothe disintegration of NPs. This might limit the efficacy of drugdelivery and absorption in the small intestine.

When increasing the degree of quaternization on TMC (TMC40 and TMC55),the stability of NPs in the pH range of 6.6-7.4 increased significantly.However, the swelling of TMC55/γ-PGA NPs at pH 7.4 was minimal (due tothe highly quaternized TMC55), which might limit the release of loadeddrugs. In contrast, TMC40/γ-PGA NPs swelled significantly withincreasing the pH value. TMC40/γ-PGA NPs (collapsed NPs or fragments)still retained a positive surface charge with a zeta potential value of17.3 mV at pH 7.4.

Thus, TMC40/γ-PGA/drug NPs have superior stability in a broader pH rangecompared to CS/γ-PGA/drug NPs. In one embodiment, at around body fluidpH of about 7.4, the bioactive nanoparticles of the present inventionmay appear to be in configuration of chitosan-shelled fragments orchitosan-containing fragments. At least a portion of the surface of thechitosan-shelled fragments or chitosan-containing fragments from thebioactive nanoparticles of the present invention shows positive zetapotential characteristics.

The results of molecular dynamic simulations showed that the molecularchains of TMC40 (in dark black) and γ-PGA (in light black) in theirself-assembled complex were tightly entangled with each other at pH 6.0.The surface of the complex was dominated by TMC40 molecules. Relaxationsof TMC40 and γ-PGA molecular chains at pH 2.5 resulted in a moderateswelling of the TMC40/γ-PGA complex, while its surface was stilldominated by the positively charged TMC molecules, thus retaining apositive surface charge.

Similarly, relaxations of TMC40 and γ-PGA molecular chains at pH 7.4resulted in a significant swelling of the TMC40/γ-PGA complex, while itssurface was still dominated by the positively charged TMC molecules,thus retaining a positive surface charge. The swollen TMC40/γ-PGA/drugnanoparticles tend to slightly disintegrate (due to the effect of its pHinstability) so to form fragments consisting of TMC40/γ-PGA/drug withsurface-dominated TMC40.

The TMC40/γ-PGA/drug fragments with surface-dominated TMC40 would adhereand infiltrate into the mucus of the epithelial membrane of theblood-brain barrier, and then trigger transiently opening the tightjunctions between enterocytes. Table 6 shows mean particle sizes, zetapotential values, and polydispersity indices of nanoparticles (NPs)self-assembled by TMC polymers with different degrees of quaternizationand γ-PGA at distinct pH environments (n=5 batches). As shown in Table6, TMC40/γ-PGA NPs still retained a positive surface charge with a zetapotential value of 17.3 mV at pH 7.4.

Freeze-Dried Nanoparticles

A pharmaceutical composition of nanoparticles of the present inventionmay comprise a first component of at least one bioactive agent, a secondcomponent of chitosan (including regular molecular weight and lowmolecular weight chitosan), and a third component that is negativelycharged. In one embodiment, the second component dominates on a surfaceof the nanoparticle. In another embodiment, the chitosan is N-trimethylchitosan.

In still another embodiment, the low molecular weight chitosan has amolecular weight less than that of a regular molecular weight chitosan.The nanoparticles may further comprise tripolyphosphate and magnesiumsulfate. For example, a first solution of (2 ml 0.1% γ-PGA aqueoussolution @pH 7.4+0.05% Insulin+0.1% Tripolyphosphate (TPP)+0.2% MgSO₄)is added to a base solution (10 ml 0.12% chitosan aqueous solution @pH6.0) as illustrated in Example no. 3 under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. The bioactive agent, the third component,tripolyphosphate and magnesium sulfate are wholly or substantiallytotally encapsulated within the nanoparticles. Supernatants werediscarded and nanoparticles were resuspended in deionized water forfreeze-drying preparation. Other operating conditions or other bioactiveagent (such as protein, peptide, siRNA, growth factor, the one definedand disclosed herein, and the like) may also apply.

Several conventional coating compounds that form a protective layer onparticles are used to physically coat or mix with the nanoparticlesbefore a freeze-drying process. The coating compounds may includetrehalose, mannitol, glycerol, and the like. Trehalose, also known asmycose, is an alpha-linked (disaccharide) sugar found extensively butnot abundantly in nature. It can be synthesized by fungi, plants andinvertebrate animals. It is associated with anhydrobiosis—the ability ofplants and animals to withstand prolonged periods of desiccation. Thesugar is thought to form a gel phase as cells dehydrate, which preventsdisruption of internal cell organelles by effectively splinting them inposition. Rehydration then allows normal cellular activity to resumewithout the major, generally lethal damage, which would normally followa dehydration/rehydration cycle. Trehalose has the added advantage ofbeing an antioxidant.

TABLE 6 Parameters of nanoparticles (NPs) self-assembled by TMC polymerswith different degrees of quaternization. Mean Particle Size ZetaPotential Polydispersity (nm) (mV) Index CS/γ-PGA NPs pH 1.2 N/A N/A 1pH 2.0 N/A N/A 1 pH 2.5 113.3 ± 1.6 38.6 ± 0.8 0.14 ± 0.01 pH 6.0 104.1± 1.2 36.2 ± 2.5 0.11 ± 0.02 pH 6.6 245.6 ± 4.5 12.9 ± 0.4 0.17 ± 0.11pH 7.0 N/A N/A 1 pH 7.4 N/A N/A 1 TMC25/γ-PGA NPs pH 1.2 N/A N/A 1 pH2.0 N/A N/A 1 pH 2.5 396.4 ± 4.7 32.1 ± 1.6 0.32 ± 0.11 pH 6.0 101.3 ±3.1 30.9 ± 2.1 0.13 ± 0.04 pH 6.6 N/A N/A 1 pH 7.0 N/A N/A 1 pH 7.4 N/AN/A 1 TMC40/γ-PGA NPs pH 1.2 N/A N/A 1 pH 2.0 N/A N/A 1 pH 2.3 272.2 ±2.3 38.6 ± 2.7 0.25 ± 0.23 pH 2.5 252.4 ± 3.5 35.4 ± 1.1 0.21 ± 0.04 pH6.0 106.3 ± 2.3 32.3 ± 2.1 0.15 ± 0.14 pH 6.6 238.3 ± 3.1 24.3 ± 1.40.09 ± 0.03 pH 7.0 296.7 ± 4.7 20.4 ± 0.3 0.18 ± 0.11 pH 7.4 498.4 ± 6.817.3 ± 0.6 0.38 ± 0.21 TMC55/γ-PGA NPs pH 1.2 N/A N/A 1 pH 2.0 252.5 ±4.1 35.6 ± 4.2 0.16 ± 0.08 pH 2.5 221.4 ± 3.5 32.5 ± 3.4 0.15 ± 0.02 pH6.0 114.6 ± 2.3 30.6 ± 3.8 0.12 ± 0.03 pH 6.6 141.2 ± 1.6 24.8 ± 3.40.15 ± 0.02 pH 7.0 144.6 ± 4.8 20.4 ± 1.7 0.18 ± 0.14 pH 7.4 141.2 ± 0.918.9 ± 4.1 0.11 ± 0.11 N/A: Precipitation of aggregates was observed.

Trehaloze has a chemical formula as C₁₂H₂₂O₁₁.2H₂O. It is listed as CASno. 99-20-7 and PubChem 7427. The molecular structure for trehalose isshown below.

Trehalose was first isolated from the ergot of rye. Trehalose is anon-reducing sugar formed from two glucose units joined by a 1-1 alphabond, giving it the name α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside.The bonding makes trehalose very resistant to acid hydrolysis, andtherefore stable in a solution at high temperatures, even under acidicconditions. The bonding also keeps non-reducing sugars in a closed-ringform, such that the aldehyde or ketone end-groups do not bind to thelysine or arginine residues of proteins (a process called glycation).Trehalose has about 45% the sweetness of sucrose. Trehalose is lesssoluble than sucrose, except at high temperatures (>80° C.). Trehaloseforms a rhomboid crystal as the dihydrate, and has 90% of the calorificcontent of sucrose in that form. Anhydrous forms of trehalose readilyregain moisture to form the dihydrate. Trehalose has also been used inat least one biopharmaceutical formulation, the monoclonal antibodytrastuzumab, marketed as Herceptin. It has a solubility of 68.9 g/100 gH₂O at 20° C.

Mannitol or hexan-1,2,3,4,5,6-hexyl (C₆H₈(OH)₆) is an osmotic diureticagent and a weak renal vasodilator. Chemically, mannitol is a sugaralcohol, or a polyol; it is similar to xylitol or sorbitol. However,mannitol has a tendency to lose a hydrogen ion in aqueous solutions,which causes the solution to become acidic. For this reason, it is notuncommon to add a substance to adjust its pH, such as sodiumbicarbonate. Mannitol has a chemical formula C₆H₁₄O₆. It is listed asCAS no. 69-65-8 and PubChem 453. The molecular structure for mannitol isshown below.

Glycerol is a chemical compound with the formula HOCH₂CH(OH)CH₂OH. Thiscolorless, odorless, viscous liquid is widely used in pharmaceuticalformulations. Commonly called glycerin or glycerine, it is a sugaralcohol and is fittingly sweet-tasting with low toxicity. Glycerol hasthree hydrophilic alcoholic hydroxyl groups that are responsible for itssolubility in water and its hygroscopic nature. Glycerol has a chemicalformula as C₃H₅(OH)₃. It is listed as CAS no. 56-81-5. The molecularstructure for glycerol is shown below.

Example No. 18 Freeze-Drying Process for Nanoparticles

Each nanoparticles (at 2.5% concentration) were mixed with a solution offour types of liquid at a 1:1 volume ratio for about 30 minutes untilfully dispersed. The mixed particle-liquid was then freeze-dried under alyophilization condition, for example, at about −80° C. and <25 mmHgpressure for about 6 hours. The parameters in a selected lyophilizationcondition may vary slightly from the aforementioned numbers. The fourtypes of liquid used in the experiment include: (A) DI water; (B)trehalose; (C) mannitol; and (D) glycerol, whereas the concentration ofthe liquid (A) to liquid (C) in the solution was set at 2.5%, 5% and10%. After a freeze-drying process, the mixed particle-liquid wasrehydrated with DI water at a 1:5 volume ratio to assess the integrityof nanoparticles in each type of liquid. The results are shown in Table7. By comparing the particle size, polydispersity index, andzeta-potential data, only the nanoparticles from the freeze-driedparticle-trehalose runs (at 2.5%, 5%, and 10% concentration level) showcomparable properties to those of the pre-lyophilization nanoparticles.Under the same data analysis, the nanoparticles from the freeze-driedparticle-mannitol runs (at 2.5%, and 5% concentration level) showsomewhat comparable properties to those of the pre-lyophilizationnanoparticles.

FIG. 16 shows an illustrative mechanism of nanoparticles released fromthe enteric-coated capsules. FIG. 16(A) shows the phase wherenanoparticles are in the gastric cavity, wherein the freeze-driednanoparticles 82 are encapsulated within an initial enteric coating orcoated capsule 81. FIG. 16(B) shows a schematic of the nanoparticlesduring the phase of entering small intestine, wherein the enteric coatand its associated capsule starts to dissolve 83 and a portion ofnanoparticles 82 is released from the capsule and contacts fluid. FIG.16(C) shows the phase of nanoparticles in the intestinal tract, whereinthe nanoparticles revert to a wet state having chitosan at its surface.In an alternate embodiment, nanoparticles may be released fromalginate-calcium coating. In preparation, nanoparticles are firstsuspended in a solution that contains calcium chloride, wherein thecalcium ions are positively charged. With a pipette, alginate withnegatively charged carboxyl groups is slowly added to the calciumchloride solution. Under gentle stirring, the alginate-calcium starts toconjugate, gel, and coat on the nanoparticle surface. In simulated oraladministration of the alginate-calcium coated nanoparticles,nanoparticles start to separate from the coating when they enter thesmall intestines.

TABLE 7 Properties of nanoparticles before and after an exemplaryfreeze-drying process. (Table 7A: before a freeze-drying process NPssolution Conc. 2.50% Size (mm) 266 Kegs 352.2 PI 0.291 Zeta Potential25.3 (Table 7B after a freeze-drying process) A: DI Water B: TrehaloseC: Mannitol D: Glycerol DI water + NPs Trehalose + NPs Mannitol + NPsGlycerol + NPs (volume 1:1) (volume 1:1) (volume 1:1) (volume 1:1) Conc.2.50% 5.00% 10.00% 2.50% 5.00% 2.50% 5.00% 10.00% Size (mm) 9229.1 302.4316.7 318.9 420.1 487.5 6449.1 7790.3 1310.5 Kcps 465.3 363.7 327.7352.2 305.4 303.7 796.1 356.1 493.3 PI 1 0.361 0.311 0.266 0.467 0.651 11 1 Zeta 25.6 24.6 24.7 24.4 25.3 Potential

Example No. 19 Freeze-Dried Nanoparticles in Animal Evaluation

In the in vivo study, rats as prepared and conditioned according toExample no. 10 were used in this evaluation. In the animal evaluationstudy, diabetic rats fasted for 12 hours and were then subjected tothree different conditions: (a) oral deionized water (DI) administrationas negative control; (b) oral insulin-loaded lyophilized nanoparticlesadministration, whereas the nanoparticles have an insulin loadingcontent of 4.4% with an insulin loading efficiency of 48.6% and areloaded in a capsule with a surface enteric coating; and (c) subcutaneous(SC) insulin injection at 5 U/kg as positive control. The blood glucoseconcentration from rat's tail was measured over time.

FIG. 19 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for oral DI administration (control) over a time interval of 10hours appears relatively constant within the experimental measurementerror range. As anticipated, the glucose decrease for the SC insulininjection method is evident in rat blood at a very early time intervalstarts to taper off after 2 hours, and ends at about 6 hours in thisexemplary study. The most important observation of the study comes fromthe oral administration route with insulin-loaded lyophilized (namely,freeze-dried) nanoparticles. Nanoparticles of this example have aninsulin LC at 4.4%, whereas nanoparticles from Example no. 10 had aninsulin LC at 14.1% in FIG. 14). With the same amount of nanoparticlesin both examples, the insulin-feeding ratio of Example no. 19 to Exampleno. 10 is about 1:3. In other words, the insulin fed to a rat in thisstudy from nanoparticles is about ⅓ of the insulin from nanoparticlesfed to rats in Example no. 10.

The blood glucose begins to decrease from the base line at about 3 hoursafter administration and sustains a lower glucose level for more than 10hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of an animal subject comprising orallyadministering insulin-containing nanoparticles with a dosage effectiveamount of the insulin to treat the diabetes, wherein at least a portionof the nanoparticles comprises a positively charged shell substrate anda negatively charged core substrate. In one embodiment, the dosageeffective amount of the insulin to treat diabetes comprises an insulinamount of between about 15 units to 45 units per kilogram body weight ofthe animal subject, preferably 20 to 40 units, and most preferably atabout 25 to 35 units insulin per kilogram body weight. In oneembodiment, the lyophilized nanoparticles may be fed as-is to an animalwithout being loaded in an enterically coated capsule.

It is known that Zn (zinc) is usually added in the biosynthesis andstorage of insulin. FIGS. 17 and 18 show a schematic of insulinconjugated with a polyanionic compound (i.e., γ-PGA in this case) viaZn, resulting in an increase in its loading efficiency and loadingcontent in the nanoparticles of the present invention. It is furtherdemonstrated that Zn may complex with the histidine and glutamic acidresidues in insulin to increase the insulin stability and enhancecontrolled release capability or sustained therapy. Some aspects of theinvention relate to a nanoparticle that enhances epithelial permeation,the nanoparticle comprising a first component of at least one bioactiveagent, a second component of low molecular weight chitosan, and a thirdcomponent that is negatively charged, wherein a stabilizer is added tostabilize the at least one bioactive agent to the negatively chargedthird component. In one embodiment, the stabilizer is zinc or calcium.

Example No. 20 Nanoparticles Loaded with Enhanced Insulin Loading

In a co-pending application, U.S. patent application Ser. No. 11/881,185filed Jul. 26, 2007, entire contents of which are incorporated herein byreference, it is disclosed that a novel nanoparticle may comprise ashell substrate of chitosan and a core substrate consisting of at leastone bioactive agent, MgSO₄, TPP, and a negatively charged substrate thatis neutralized with chitosan in the core. FIG. 20 shows insulin-loadednanoparticles with a core composition comprised of γ-PGA, MgSO₄, sodiumtripolyphosphate (TPP), and insulin. Nanoparticles were obtained uponaddition of core component, using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a CS aqueous solution (pH 6.0,10 ml) at certain concentrations under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Supernatants were discarded and nanoparticleswere resuspended in deionized water for further studies. In oneembodiment, nanoparticles are encapsulated in a gelcap, or arelyophilized before being loaded in a gelcap or in a tablet. Thenanoparticles thus obtained via the simple and mild ionic-gelationmethod described herein show typical characteristics in a spheroidalconfiguration with a particle size of between about 50 to 400 nm, apositive surface charge and a narrow polydispersity index. The sodiumtripolyphosphate has a chemical formula of Na₅P₃O₁₀ as shown below:

In the example, the core composition may be varied and evaluated with apreferred composition of 2 ml γ-PGA aqueous solution at pH 7.4 withinsulin, MgSO₄ and TPP, resulting in a ratio ofCS:γ-PGA:TPP:MgSO4:insulin=6.0:1.0:1.0:2.0:0.05. Thus, the nanoparticlesshow characteristics as disclosed herein with a chitosan shell and acore composition consisting of γ-PGA, MgSO₄, TPP, and insulin and havean average loading efficiency of 72.8% insulin and an average loadingcontent of 21.6% insulin.

In the enhanced drug loading of the present example, there provides twoor more distinct ionic crosslink mechanisms. In one embodiment, thenanoparticles of the present invention may have a structure or matrix ofinterpenetrated ionic-crosslinks (that is, elongate ionic-crosslinkchains) including a first ionic-crosslink chain of NH₃ ⁺ of CS with COO⁻of γ-PGA, a second ionic-crosslink chain of NH₃ ⁺ of CS with SO₄ ²⁻ ofMgSO₄, a third ionic-crosslink chain of Mg²⁺ of MgSO₄ with COO⁻ ofγ-PGA, and/or a fourth ionic-crosslink chain of Na₃P₃O₁₀ ²⁻ of TPP withNH₃ ⁺ of CS or Mg²⁺ of MgSO₄.

Some aspects of the invention relate to a nanoparticle composition fororal administration with an insulin loading efficiency and content athigher than 45% and 14% (preferably up to about 73% and 22%),respectively. The prepared nanoparticles (NPs) are stable at a pH rangeof 2.0 to 7.1. This broad range allows the chitosan-shelled nanoparticleto be temporarily stable in most of the intestine region (includingduodenum, jejunum, and ileum) for enhanced membrane absorption andpermeability of active ingredient (for example, insulin, exenatide orpramlintide). Some aspects of the invention provide a chitosan-shellednanoparticle with a core composition of γ-PGA, MgSO₄, TPP, and at leastone bioactive agent, such as insulin, exenatide, or pramlintide fortreatment of diabetes. In an alternate embodiment, some aspects of theinvention provide a chitosan-shelled nanoparticle with a corecomposition consisted of γ-PGA, MgSO₄, TPP, and at least one bioactiveagent. In one embodiment, negatively charged γ-PGA may conveniently besubstituted by another negatively charge substrate, such as heparin. Inan experiment following the experimental conditions of Example no. 20 bysubstituting insulin with exenatide, chitosan-shelled nanoparticles witha core composition of γ-PGA, MgSO₄, TPP, and exenatide exhibit similarphysical and mechanical properties compared to the ones with insulin.

FIG. 21 shows an in vivo subcutaneous study using insulin injectablesand insulin-containing nanoparticles. The insulin-containingnanoparticles exhibit different pharmacodynamics and/or pharmacokineticsin a sustained releasing manner. Some aspects of the invention relate toa pharmaceutical composition of nanoparticles for subcutaneous or bloodvessel administration in an animal subject, the nanoparticles comprisinga shell portion that is dominated by positively charged chitosan, a coreportion that contains negatively charged substrate, wherein thenegatively charged substrate is at least partially neutralized with aportion of the positively charged chitosan in the core portion, and atleast one bioactive agent loaded within the nanoparticles.

Some aspects of the invention relate to a method of delivering abioactive agent to blood circulation in an animal subject, comprising:(a) providing nanoparticles according to a preferred embodiment of thepharmaceutical composition of the present invention, wherein thenanoparticles are formed via a simple and mild ionic-gelation method;(b) administering the nanoparticles orally toward the intestine of theanimal subject via stomach; (c) urging the nanoparticles to be absorbedonto a surface of an epithelial membrane of the intestine viamuco-adhesive chitosan-shelled nanoparticles; (d) permeating bioactiveagent to pass through an epithelial barrier of the intestine; and (e)releasing the bioactive agent into the blood circulation. In oneembodiment, the bioactive agent is selected from the group consisting ofexenatide, pramlintide, insulin, insulin analog, and combinationsthereof. In another embodiment, the bioactive agent permeates throughthe tight junctions of the epithelial membrane when chitosan-shellednanoparticles break up and release the bioactive agent at vicinity ofthe tight junctions.

Some aspects of the invention relate to a method for inducing aredistribution of tight junctions' ZO-1 protein, leading to atranslocation of the ZO-1 protein to the cytoskeleton that accompaniesincreased permeation in an animal subject, the method comprisingadministering bioactive nanoparticles into the animal subject with aneffective dosage to induce the redistribution, wherein the bioactivenanoparticles comprise a shell substrate of chitosan and a coresubstrate that comprises poly(glutamic acid) and the bioactive agentthat is selected from the group consisting of exenatide, pramlintide,insulin, insulin analog, and combinations thereof.

Nanoparticles Loaded with Tumor Necrosis Factor Inhibitors

Tumor necrosis factor (TNF) promotes an inflammatory response, which inturn causes many of the clinical problems associated with autoimmunedisorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn'sdisease, psoriasis, and refractory asthma. These disorders are sometimestreated by using a TNF inhibitor. This inhibition can be achieved with amonoclonal antibody such as infliximab (Remicade) or adalimumab(Humira), or with a circulating receptor fusion protein such asetanercept (Enbrel). Another example is pentoxifylline.

This potential applicability of anti-TNF therapies in the treatment ofrheumatoid arthritis (RA) is based on the recognition of the role ofTNF-alpha is the “master regulator” of the inflammatory response in manyorgan systems. TNF and the effects of TNF are also inhibited by a numberof natural compounds, including curcumin (a compound present inturmeric) and catechins (in green tea). Tumor necrosis factor-alpha(TNFα) is a cytokine produced by monocytes and macrophages, two types ofwhite blood cells. It mediates the immune response by increasing thetransport of white blood cells to sites of inflammation, and throughadditional molecular mechanisms which initiate and amplify inflammation.

Adalimumab (brand name HUMIRA) is a TNF inhibitor, after infliximab andetanercept, to be approved in the United States. Like infliximab andetanercept, adalimumab binds to TNFα, preventing it from activating TNFreceptors. Adalimumab is constructed from a fully human monoclonalantibody, while infliximab is a mouse-human chimeric antibody, andetanercept is a TNF receptor-IgG fusion protein. TNFα inactivation hasproven to be important in downregulating the inflammatory reactionsassociated with autoimmune diseases. As of 2008, adalimumab has beenapproved by the FDA for the treatment of rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, moderate to severechronic psoriasis, and juvenile idiopathic arthritis.

Adalimumab has a chemical formula of C₆₄₂₈H₉₉₁₂N₁₆₉₄O₁₉₈₇S₄₆ and amolecular mass of 144190.3 g/mol. Humira (brand name is an abbreviationof “Human Monoclonal Antibody in Rheumatoid Arthritis”) is marketed as asubcutaneously injected treatment, typically by the patient at home. Itcannot be administered orally, because the digestive system woulddestroy the drug in its current state, unless the drug is encapsulatedin nanoparticles of the present invention.

Etanercept (Enbrel) is a recombinant-DNA drug made by combining twoproteins (a fusion protein). It links human soluble TNF receptor to theFc component of human immunoglobulin G1 (IgG1) and acts as a TNFinhibitor. Etanercept has a chemical formula of C₂₂₂₄H₃₄₇₅N₆₂₁O₆₉₈S₃₆and a molecular mass of 51234.9 g/mol. Etanercept binds to TNFα anddecreases its role in disorders involving excess inflammation in humansand other animals, including autoimmune diseases such as ankylosingspondylitis, juvenile rheumatoid arthritis, psoriasis, psoriaticarthritis, rheumatoid arthritis, and, potentially a variety of otherdisorders mediated by excess TNFα.

Infliximab (brand name Remicade) is a drug used to treat autoimmunedisorders. Infliximab is known as a “chimeric monoclonal antibody” (theterm “chimeric” refers to the use of both mouse (murine) and humancomponents of the drug (i.e. murine binding F_(ab) domains and humanconstant F_(c) domains). The drug blocks the action of the pleiotropicproinflammatory TNFα (tumor necrosis factor alpha) by binding to it andpreventing it from signaling the receptors for TNFα on the surface ofcells. TNFα is one of the key cytokines that triggers and sustains theinflammation response.

Remicade is administered by intravenous infusion, typically at 6-8 weekintervals, and at a clinic or hospital. It cannot be administeredorally, because the digestive system would destroy the drug unless it isencapsulated in nanoparticles of the present invention. Infliximabneutralizes the biological activity of TNFα by binding with highaffinity to the soluble (free floating in the blood) and transmembrane(located on the outer membranes of T cells and similar immune cells)forms of TNFα and inhibits or prevents the effective binding of TNFαwith its receptors.

Remicade and Humira (another TNF antagonist) are in the subclass of“anti-TNF antibodies” (they are in the form of naturally occurringantibodies), and are capable of neutralizing all forms (extracellular,transmembrane, and receptor-bound) of TNF alpha. Enbrel, a third TNFantagonist, is in a different subclass (receptor-construct fusionprotein), and, because of its modified form, cannot neutralizereceptor-bound TNFa. Additionally, the anti-TNF antibodies Humira andRemicade have the capability of lysing cells involved in theinflammatory process, whereas the receptor fusion protein apparentlylacks this capability. Although the clinical significance of thesedifferences has not been absolutely proven, they may account for thediscrepancies of these drugs in both efficacy and side effects.

Infliximab has high specificity for TNFα, and does not neutralize TNFbeta (TNFβ, also called lymphotoxin α), a related but less inflammatorycytokine that utilizes the same receptors as TNFα. Biological activitiesthat are attributed to TNFα include: induction of proinflammatorycytokines such as interleukin (IL-1 and IL-6), enhancement of leukocytemovement or migration from the blood vessels into the tissues byincreasing the permeability of endothelial layer of blood vessels; andincreasing the release of adhesion molecules. Infliximab preventsdisease in transgenic mice (a special type of mice that are biologicallyengineered to produce a human form of TNFα and are used to test thesedrugs for results that might be expected in humans). These experimentalmice develop arthritis as a result of their production of human TNFα,and when administered after disease onset, infliximab allows erodedjoints to heal.

Infliximab has a chemical formula C₆₄₂₈H₉₉₁₂N₁₆₉₄O₁₉₈₇S₄₆ and amolecular mass of 144190.3 g/mol. REMICADE (infliximab) is an advancedtreatment that has been shown to have substantial benefits in patientswith a number of inflammatory disorders involving the immune system.REMICADE targets specific proteins in the body's immune system to helpcontrol the development of inflammation, significantly reducing painfulsymptoms in diseases such as plaque psoriasis, rheumatoid arthritis,psoriatic arthritis, adult Crohn's disease, pediatric Crohn's disease,ulcerative colitis, and ankylosing spondylitis.

REMICADE is a type of protein that recognizes, attaches to, and blocksthe action of a substance in the body called tumor necrosis factor(TNF). TNF is made by certain blood cells in the body. REMICADE will notcure plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, adultCrohn's disease, pediatric Crohn's disease, ulcerative colitis, andankylosing spondylitis, but blocking TNF may reduce the inflammationcaused in the body.

Some aspects of the invention relate to a method of reducinginflammatory response caused by tumor necrosis factor in an animalsubject, the method comprising orally administering nanoparticlescomposed of a TNF inhibitor, chitosan, and a core substrate ofpoly(glutamic acid) or heparin. In one embodiment, the inhibitor can bea monoclonal antibody such as infliximab (Remicade) or adalimumab(Humira), or a circulating receptor fusion protein such as etanercept(Enbrel). In one embodiment, a TNF inhibitor nanoparticle formulationconsisting of at least one inhibitor selected from the group consistingof infliximab, adalimumab and etanercept, chitosan, and one corenegatively charged substrate of poly(glutamic acid) or heparin.

Nanoparticles Loaded with Bacteriophage

A bacteriophage is any one of a number of viruses that infect bacteria.The term is commonly used in its shortened form, phage. Typically,bacteriophages consist of an outer protein hull enclosing geneticmaterial. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA(‘ss-’ or ‘ds-’ prefix denotes single strand or double strand) between 5and 500 kilo nucleotides long with either circular or lineararrangement. Bacteriophages are much smaller than the bacteria theydestroy—usually between 20 and 200 nm in size.

Phages are estimated to be the most widely distributed and diverseentities in the biosphere. Phages are ubiquitous and can be found in allreservoirs populated by bacterial hosts, such as soil or the intestinesof animals. One of the densest natural sources for phages and otherviruses is sea water, where up to 9×10⁸ virions per milliliter have beenfound in microbial mats at the surface, and up to 70% of marine bacteriamay be infected by phages. They have been used for over 60 years as analternative to antibiotics in the former Soviet Union and EasternEurope. They are seen as a possible therapy against multi drug resistantstrains of many bacteria.

To enter a host cell, bacteriophages attach to specific receptors on thesurface of bacteria, including lipopolysaccharides, teichoic acids,proteins, or even flagella. This specificity means that a bacteriophagecan only infect certain bacteria bearing receptors that they can bindto, which in turn determines the phage's host range. As phage virions donot move independently, they must rely on random encounters with theright receptors when in a solution (blood, lymphatic circulation,irrigation, soil water etc.).

Complex bacteriophages use a syringe-like motion to inject their geneticmaterial into the cell. After making contact with the appropriatereceptor, the tail fibers bring the base plate closer to the surface ofthe cell. Once attached completely, the tail contracts, possibly withthe help of ATP present in the tail, injecting genetic material throughthe bacterial membrane.

Within minutes, bacterial ribosomes start translating viral mRNA intoprotein. For RNA-based phages, RNA replicase is synthesized early in theprocess. Proteins modify the bacterial RNA polymerase so that itpreferentially transcribes viral mRNA. The host's normal synthesis ofproteins and nucleic acids is disrupted, and it is forced to manufactureviral products instead. These products go on to become part of newvirions within the cell, helper proteins which help assemble the newvirions, or proteins involved in cell lysis.

Phages may be released via cell lysis, by extrusion, or, in a few cases,by budding. Lysis, by tailed phages, is achieved by an enzyme calledendolysin which attacks and breaks down the cell wall peptidoglycan. Analtogether different phage type, the filamentous phages, causes the hostcell to continually secrete new virus particles. Released virions aredescribed as free and unless defective are capable of infecting a newbacterium. Budding is associated with certain Mycoplasma phages. Incontrast to virion release, phages displaying a lysogenic cycle do notkill the host but rather become long-term residents as prophage.

In August, 2006 the United States Food and Drug Administration (FDA)approved using bacteriophages on cheese to kill the Listeriamonocytogenes bacteria, giving them GRAS status (Generally Recognized AsSafe). In July 2007, the same bacteriophages were approved for use onall food products.

Some aspects of the invention relate to a method of mitigating bacteriain an animal subject, the method comprising orally administeringnanoparticles composed of at least one bacteriophage, chitosan, and acore substrate of poly(glutamic acid) or heparin. One aspect of theinvention relates to a bactericide nanoparticle formulation comprisingat least one bacteriophage, chitosan, and a core substrate ofpoly(glutamic acid) or heparin. In one embodiment, a bactericidenanoparticle formulation consisting of at least one bacteriophage,chitosan, and one core negatively charged substrate of poly(glutamicacid) or heparin. In a further embodiment, the nanoparticles areencapsulated in capsules, wherein the capsules may be treated withenteric coating. In one embodiment, the capsules further comprise apermeation enhancer, wherein the permeation enhancer is selected fromthe group consisting of Ca²⁺ chelators, bile salts, anionic surfactants,medium-chain fatty acids, phosphate esters, chitosan, and chitosanderivatives. In another embodiment, the capsule may contain solubilizersuch as GRAS or other pharmacopoeial excipients.

Example No. 21 Nanoparticles Loaded with Pemetrexed

Pemetrexed (brand name Alimta®) is a chemotherapy drug manufactured andmarketed by Eli Lilly and Company. Its indications are the treatment ofpleural mesothelioma as well as non-small cell lung cancer. Pemetrexedhas a systematic (IUPAC) name2-[4-[2-(4-amino-2-oxo-3,5,7-triazabicyclo[4.3.0]nona-3,8,10-trien-9-yl)ethyl]benzoyl]aminopentanedioicacid, a chemical formula C₂₀H₂₁N₅O₆ and a molecular mass of 427.411g/mol. Pemetrexed is chemically similar to folic acid and is in theclass of chemotherapy drugs called folate antimetabolites. It works byinhibiting three enzymes used in purine and pyrimidinesynthesis—thymidylate synthase (TS), dihydrofolate reductase (DHFR), andglycinamide ribonucleotide formyltransferase (GARFT). By inhibiting theformation of precursor purine and pyrimidine nucleotides, pemetrexedprevents the formation of DNA and RNA, which are required for the growthand survival of both normal cells and cancer cells. In February 2004,the Food and Drug Administration approved pemetrexed for treatment ofmalignant pleural mesothelioma, a type of tumor of the lining of thelung, in combination with cisplatin. In September 2008, the FDA grantedapproval as a first-line treatment, in combination with cisplatin,against of locally-advanced and metastatic non-small cell lung cancer,or NSCLC, in patients with non-squamous histology. Trials are currentlytesting it against esophagus and other cancers.

In one exemplary preparation, nanoparticles were obtained upon additionof a mixture of γ-PGA plus pemetrexed aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrationshigher than 0.10% by w/v under magnetic stirring at room temperature toensure positive surface charge. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Pemetrexed is wholly orsubstantially totally encapsulated within the nanoparticles.Supernatants were discarded and nanoparticles were resuspended indeionized water as the solution products, further encapsulated incapsules/enteric capsules or further treated with lyophilizationfreeze-dried process. The nanoparticles thus obtained via the simple andmild ionic-gelation method described herein show typical characteristicsin a spheroidal configuration with a particle size of between about 50to 400 nm, a positive surface charge and a narrow polydispersity index.

Epirubicin is an anthracycline drug used for chemotherapy. It ismarketed by Pfizer under the trade name Ellence in the U.S. andPharmorubicin or Epirubicin elsewhere. Similarly to otheranthracyclines, epirubicin acts by intercalating DNA strands.Intercalation results in complex formation that inhibits DNA and RNAsynthesis. It also triggers DNA cleavage by topoisomerase II, resultingin mechanisms that lead to cell death. Binding to cell membranes andplasma proteins may be involved in the compound's cytotoxic effects.Epirubicin also generates free radicals that cause cell and DNA damage.Epirubicin is favored over doxorubicin, the most popular anthracycline,in some chemotherapy regimens as it appears to cause fewer side-effects.Epirubicin has a different spatial orientation of the hydroxyl group atthe 4′ carbon of the sugar, which may account for its faster eliminationand reduced toxicity. Epirubicin is primarily used against breast andovarian cancer, gastric cancer, lung cancer, and lymphomas. Itssystematic (IUPAC) name is10-(4-amino-5-hydroxy-6-methyl-oxan-2-yl)oxy-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-9,10-dihydro-7H-tetracene-5,12-dione.The chemical formula is C₂₇H₂₉NO₁₁ with a molecular mass 543.519 g/mol.

Irinotecan is a chemotherapy agent that is a topoisomerase 1 inhibitor.Chemically, it is a semisynthetic analogue of the natural alkaloidcamptothecin. Its main use is in colon cancer, particularly incombination with other chemotherapy agents. Irinotecan was firstintroduced in Japan by the Pharmaceutical arm of Yakult Honsha asCampto. In 1994, it received accelerated FDA approval in the UnitedStates, where it is now marketed by Pfizer as Camptosar. It is alsoknown as CPT-11. Its systematic (IUPAC) name is(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′,4′bipiperidine]-1′-carboxylate. It has a chemical formula C₃₃H₃₈N₄O₆ witha molecular mass 677.185 g/mol (hydrochloride). Irinotecan is activatedby hydrolysis to SN-38, an inhibitor of topoisomerase I. This is theninactivated by glucuronidation by uridine diphosphateglucoronosyltransferase 1A1 (UGT1A1). The inhibition of topoisomerase Iby the active metabolite SN-38 eventually leads to inhibition of bothDNA replication and transcription.

Example No. 22 Nanoparticles Loaded with Gemcitabine

Gemcitabine is a nucleoside analog used as chemotherapy. It is marketedas Gemzar® by Eli Lilly and Company. Gemcitabine has a systematic(IUPAC) name as4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidin-2-one.Gemcitabine has a chemical formula C₉H₁₁F₂N₃O₄ and a molecular mass of263.198 g/mol. Gemcitabine is used in various carcinomas: non-small celllung cancer, pancreatic cancer, bladder cancer and breast cancer. It isbeing investigated for use in oesophageal cancer, and is usedexperimentally in lymphomas and various other tumor types. Gemcitabinerepresents an advance in pancreatic cancer care. It is also not asdebilitating as other forms of chemotherapy. A study reported in theJournal of the American Medical Association suggested that gemcitabineshows benefit in patients with pancreatic cancer who were considered tohave successful tumor resections. GemCarbo chemotherapy (consisting ofgemcitabine, as known as Gemzar and Carboplatin, which are bothcolourless fluids) is used to treat several different types of cancer,but most commonly lung cancer.

In one exemplary preparation, nanoparticles were obtained upon additionof a mixture of γ-PGA and gemcitabine aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrationshigher than 0.10% by w/v under magnetic stirring at room temperature toensure positive surface charge. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Gemcitabine is wholly orsubstantially totally encapsulated within the nanoparticles.Supernatants were discarded and nanoparticles were resuspended indeionized water as the solution products, further encapsulated incapsules/enteric capsules or treated with lyophilization freeze-driedprocess. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge, and a narrow polydispersity index.

Example No. 23 Nanoparticles Loaded with Drotrecogin Alfa (Activated)

Drotrecogin alfa (activated) (Xigris®, marketed by Eli Lilly andCompany) is a recombinant form of human activated protein C that hasanti-thrombotic, anti-inflammatory, and profibrinolytic properties.Drotrecogin alpha (activated) belongs to the class of serine proteases.It is used mainly in intensive care medicine as a treatment for severesepsis. Drotrecogin alpha (activated) has a systematic (IUPAC) name asActivated human protein C; it has a chemical formula C₁₇₈₆H₂₇₇₉N₅₀₉ ^(O)₅₁₉S₂₉ and a molecular mass of 55000 g/mol. The specific mechanism bywhich drotrecogin exerts its effect on survival in patients with severesepsis is not completely understood. In vitro data suggest thatactivated protein C exerts an antithrombotic effect by inhibitingfactors Va and VIIIa, and that it has indirect profibrinolytic activityby inhibiting plasminogen activator inhibitor-1 (PAI-1). In vitro dataalso suggest that activated protein C may exert an anti-inflammatoryeffect by inhibiting tumor necrosis factor production, by blockingleukocyte adhesion to selectins, and by limiting the thrombin-inducedinflammatory responses within the microvascular endothelium. If the i.v.dosage guidelines are followed, the drug reaches peak plasma levelsafter two hours and is completely cleared from plasma two hours afterthe termination of the infusion period. Endogenous plasma proteaseinhibitors deactivate drotrecogin. Therefore, no dosage adjustment isneeded in elderly patients, or in patients with renal or hepaticdysfunction. Drotrecogin is indicated for the reduction of mortality inadult patients with severe sepsis (sepsis associated with acute organdysfunction) who have a high risk of death.

In one exemplary preparation, nanoparticles were obtained upon additionof a mixture of γ-PGA and drotrecogin alpha (activated) aqueous solution(2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech ScientificInc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Drotrecoginalpha (activated) is wholly or substantially totally encapsulated withinthe nanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products. Then they wereeither encapsulated in capsules/enteric capsules or treated withlyophilization freeze-dried process. The nanoparticles thus obtained viathe simple and mild ionic-gelation method described herein show typicalcharacteristics in a spheroidal configuration with a particle size ofbetween about 50 to 400 nm, a positive surface charge and a narrowpolydispersity index.

Factor IX (or Christmas factor) is one of the serine proteases of thecoagulation system; it belongs to peptidase family S1. Deficiency ofthis protein causes hemophilia B. Factor IX is inactive unless activatedby factor XIa (of the contact pathway) or factor VIIa (of the tissuefactor pathway). When activated into factor IXa, it acts by hydrolysingone arginine-isoleucine bond in factor X to form factor Xa. It requirescalcium, membrane phospholipids, and factor VIII as cofactors to do so.Factor IX complex is currently via injectable routes. Its generic nameis Factor IX, human recombinant-injection. In use, Factor IX is a partof blood needed for clotting which stops bleeding. Persons with lowFactor levels are at risk for bleeding. This medication is used toprevent or control bleeding episodes in persons with low Factor levels(hemophilia, Christmas disease). It is also used to reverse the effectsof warfarin blood thinner. Factor IX has been incorporated in thenanoparticle formulation via the simple and mild ionic-gelation methoddescribed herein, the nanoparticles thus obtained show typicalcharacteristics in a spheroidal configuration with a particle size ofbetween about 50 to 400 nm, a positive surface charge and a narrowpolydispersity index.

Example No. 24 Nanoparticles Loaded with Humatrope

Humatrope® (somatotropin or somatropin) is a polypeptide hormone of rDNAorigin. Manufactured by Eli Lilly and Company, it is used to stimulatelinear growth in pediatric patients who lack adequate normal humangrowth hormone. It has 191 amino acid residues and a molecular weight of22,125 daltons. Its amino acid sequence is identical to that of humangrowth hormone of pituitary origin (anterior lobe). Humatrope issynthesized in a strain of E. coli modified by the addition of a genefor human growth hormone. Other human growth hormone produced from rDNAsinclude; Omnitrope® (Sandoz), Nutropin, Norditropin, Genotropin®(Pfizer).

In one exemplary preparation, nanoparticles were obtained upon additionof a mixture of γ-PGA and Humatrope aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrationshigher than 0.10% by w/v under magnetic stirring at room temperature toensure positive surface charge. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Humatrope is wholly orsubstantially totally encapsulated within the nanoparticles.Supernatants were discarded and nanoparticles were resuspended indeionized water as the solution products, further encapsulated incapsules/enteric capsules or treated with lyophilization freeze-driedprocess. The nanoparticles thus obtained via the simple and mildionic-gelation method described herein show typical characteristics in aspheroidal configuration with a particle size of between about 50 to 400nm, a positive surface charge, and a narrow polydispersity index.

The major isoform of the human growth hormone (GH) is a protein of 191amino acids and a molecular weight of 22124 daltons. The structureincludes four helices necessary for functional interaction with the GHreceptor. GH is structurally and apparently evolutionarily homologous toprolactin and chorionic somatomammotropin. Despite marked structuralsimilarities between growth hormone from different species, only humanand primate growth hormones have significant effects in humans. Effectsof growth hormone on the tissues of the body can generally be describedas anabolic (building up). Like most other protein hormones, GH acts byinteracting with a specific receptor on the surface of cells. In oneaspect, GH stimulating the increase in height in childhood is the mostwidely known effect of GH, and appears to be caused by at least twomechanisms. In another aspect, because polypeptide hormones are not fatsoluble, they cannot penetrate sarcolemma. Thus, GH exerts some of itseffects by binding to receptors on target cells, where it activates asecondary messenger. Through this mechanism, GH directly stimulatesdivision and multiplication of chondrocytes of cartilage. These are theprimary cells in the growing ends (epiphyses) of children's long bones.

GH also stimulates production of insulin-like growth factor 1 (IGF-1,formerly known as somatomedin C), a hormone homologous to proinsulin.The liver is a major target organ of GH for this process, and is theprincipal site of IGF-1 production. IGF-1 has growth-stimulating effectson a wide variety of tissues. Additional IGF-1 is generated withintarget tissues, making it apparently both an endocrine and anautocrine/paracrine hormone. IGF-1 also has stimulatory effects onosteoblast and chondrocyte activity to promote bone growth. In additionto increasing height in children and adolescents, growth hormone hasmany other effects on the body, such as: increasing calcium retention,and strengthening and increasing the mineralization of bone; increasingmuscle mass through sarcomere hyperplasia; promoting lipolysis;increasing protein synthesis; stimulating the growth of all internalorgans excluding the brain; playing a role in fuel homeostasis; reducingliver uptake of glucose; promoting gluconeogenesis in the liver;contributing to the maintenance and function of pancreatic islets; andstimulating the immune system.

Example No. 25 Nanoparticles Loaded with Anti-Hemophilic Factors

Hemophilia is a group of hereditary genetic disorders that impair thebody's ability to control blood clotting or coagulation. The effects ofthis sex-linked, X chromosome disorder are manifested almost entirely inmales, although the gene for the disorder is inherited from the mother.Females have two X chromosomes while males have only one, lacking a‘back up’ copy for the defective gene. Females are therefore almostexclusively carriers of the disorder, and may have inherited it fromeither their mother or father. These genetic deficiencies may lowerblood plasma clotting factor levels of coagulation factors needed for anormal clotting process. When a blood vessel is injured, a temporaryscab does form, but the missing coagulation factors prevent fibrinformation which is necessary to maintain the blood clot. Thus, ahemophiliac does not bleed more intensely than a normal person, but fora much longer amount of time. In severe hemophiliacs, even a minorinjury could result in blood loss lasting days, weeks, or may never healcompletely. The critical risk here is with normally small injuries,which, due to missing factor VIII, take extended periods of time toheal. In areas such as the brain or inside joints, this can be fatal orpermanently debilitating.

In one exemplary preparation, nanoparticles were obtained upon additionof a mixture of γ-PGA and SonoSeven (a recombinant human coagulationFactor VIIa; rFVIIa) aqueous solution (2 ml), using a pipette (0.5-5 ml,PLASTIBRAND®, BrandTech Scientific Inc., Germany), into a low-MW CSaqueous solution (pH 6.0, 10 ml) at concentrations higher than 0.10% byw/v under magnetic stirring at room temperature to ensure positivesurface charge. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. rFVIIa is wholly or substantially totallyencapsulated within the nanoparticles. Supernatants were discarded andnanoparticles were resuspended in deionized water as the solutionproducts. Then they were either encapsulated in capsules/entericcapsules or treated with lyophilization freeze-dried process. Thenanoparticles thus obtained via the simple and mild ionic-gelationmethod described herein show typical characteristics in a spheroidalconfiguration with a particle size of between about 50 to 400 nm, apositive surface charge, and a narrow polydispersity index.

Hemophilia A is an X-linked genetic disorder involving a lack offunctional clotting Factor VIII and represents 90% of hemophilia cases.Hemophilia B is an X-linked genetic disorder involving a lack offunctional clotting Factor IX. It is less severe but more uncommon thanHemophilia A. Hemophilia C is an autosomal recessive genetic disorderinvolving a lack of functional clotting Factor XI. Though there is nocure for hemophilia, it can be conventionally controlled with regularinfusions of the deficient clotting factor, i.e. factor VIII inhemophilia A or factor IX in hemophilia B. Factor replacement can beeither isolated from human blood serum, recombinant, or a combination ofthe two. Some hemophiliacs develop antibodies (inhibitors) against thereplacement factors given to them, so the amount of the factor has to beincreased or non-human replacement products must be given, such asporcine factor VIII. Inhibitors are a complication of hemophilia. Peoplewith severe Hemophilia A or B is usually treated by replacing themissing Factor VIII or Factor IX through infusion. For some people,however, this treatment does not work. Their bodies react as though thetreatment is an invader and their immune system develops antibodies(inhibitors) which attack and neutralize the Factor VIII or IX. Theneutralized factor is not able to stop the bleeding. Factor VIII is aglycoprotein procofactor.

In one aspect, Xyntha™ (Wyeth) anti-hemophilic factor (recombinant),plasma/serum-free is indicated for the control and prevention ofbleeding episodes in an animal subject with hemophilia A (congenitalfactor VIII deficiency or classic hemophilia) and for surgicalprophylaxis in an animal subject with hemophilia A. Patients can controlbleeding episodes with normal plasma, concentrates of factor VII, orgenetically produced (recombinant) factor VII. People need frequenttreatment during bleeding episodes because factor VII does not lastlong. Women can control menstrual bleeding with oral contraceptives.

An activated concentrate of factor VII called NovoSeven can also beused. In one aspect, NovoSeven® (Novo Nordisk) is a recombinant humancoagulation Factor VIIa (rFVIIa), intended for promoting hemostasis byactivating the extrinsic pathway of the coagulation cascade. NovoSevenis a vitamin k-dependent glycoprotein consisting of 406 amino acidresidues (MW 50 k Dalton). NovoSeven is structurally similar to humanplasma-derived Factor VIIa. NovoSeven is supplied as a sterile, whitelyophilized powder of rFVIIa in single-use vials. Some other brand namesfor the generic anti-hemophilic factor, recombinant-injection mayinclude: Bioclate (Aventis Behring), Helixate (CSL Behring), Kogenate(Bayer Healthcare), Recombinate (Baxter Healthcare) Advate (BaxterHealthcare), Alphanate (Grifols SA), Hemofil-M (Baxter Healthcare),Humate-P (CLS Behrng), Koate (Talecris Biotherapeutics), Monarc-M(Baxter Healthcare), Monoclate-P (CSL Behring), Refacto (Wyeth), andothers.

Idarubicin or 4-demethoxydaunorubicin is an anthracycline antileukemicdrug that is currently combined with cytosine arabinoside as a firstline treatment of acute myeloid leukemia. It belongs to the family ofdrugs called antitumor antibiotics. It is distributed under the tradenames Zavedos (UK) and Idamycin (USA).

Some aspects of the invention relate to a method of promoting hemostasiscaused by hemophilia in an animal subject, the method comprising orallyadministering nanoparticles composed of anti-hemophilic factor,chitosan, and a core substrate of poly(glutamic acid) or heparin,wherein a surface of the nanoparticles is dominated by chitosan. In oneembodiment, the oral nanoparticles comprise chitosan or chitosanderivatives as a permeation enhancer. In another embodiment, thenanoparticles further comprise a permeation enhancer.

Some aspects of the invention relate to a pharmaceutical composition fortreating a subject comprising two or more bioactive nanoparticles, thustreating the subject by co-administering the bioactive nanoparticles tothe subject, wherein a first bioactive nanoparticle comprises a shellportion that is dominated by positively charged chitosan, a core portionthat contains negatively charged substrate, wherein the negativelycharged substrate is at least partially neutralized with a portion ofthe positively charged chitosan, and at least a first bioactive agent,and wherein a second bioactive nanoparticle comprises a shell portionthat is dominated by positively charged chitosan, a core portion thatcontains negatively charged substrate, wherein the negatively chargedsubstrate is at least partially neutralized with a portion of thepositively charged chitosan, and at least a second bioactive agent.

In one embodiment, the first and second nanoparticles are loaded in samecapsules. In another embodiment, the first nanoparticle is loaded in afirst capsule and the second nanoparticle is loaded in a second capsule.In one embodiment, the capsules are treated with enteric coating. In afurther embodiment, the capsules further comprise at least a solubilizeror pharmacopoeial excipients. In another embodiment, the capsulesfurther comprise a permeation enhancer, wherein the permeation enhanceris selected from the group consisting of Ca²⁺ chelators, bile salts,anionic surfactants, medium-chain fatty acids, phosphate esters,chitosan, and chitosan derivatives. In one embodiment, the first andsecond nanoparticles are loaded in tablets or pills.

In one embodiment, the first bioactive agent of the pharmaceuticalcomposition comprises non-insulin anti-diabetic drug, wherein thenon-insulin anti-diabetic drug is selected from the group consisting ofinsulin sensitizers, insulin secretagogues, GLP-1 analogs, and DPP-4inhibitors, alpha-glucosidase inhibitors, amylin analog, sodium-glucoseco-transporter type 2 (SGLT2) inhibitors, benfluorex, and tolrestat. Inanother embodiment, the second bioactive agent of the pharmaceuticalcomposition comprises insulin or insulin analogs.

Example No. 26 Nanoparticles Loaded with DTPA

Some aspects of the invention relate to a pharmaceutical composition ofnanoparticle comprising chitosan, PGA-complexone conjugate and abioactive agent. In one embodiment, the PGA-complexone conjugate maybroadly include a conjugate with PGA derivatives such as γ-PGA, α-PGA,derivatives of PGA or salts of PGA, whereas the complexone may coverDTPA (diethylene triamine pentaacetic acid), EDTA (ethylene diaminetetra acetate), IDA (iminodiacetic acid), NTA (nitrilotriacetic acid),EGTA (ethylene glycol tetraacetic acid), BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid), NOTA(2,2′,2″-(1,4,7-triazonane-1,4,7-triyl)triacetic acid), and the like. Apolyamino carboxylic acid (complexone) is a compound containing one ormore nitrogen atoms connected through carbon atoms to one or morecarboxyl groups.

Diethylene triamine pentaacetic acid (DTPA) is a polyamino carboxylicacid consisting of a diethylenetriamine backbone modified with fivecarboxymethyl groups. The molecule can be viewed as an expanded versionof EDTA. DTPA is used as its conjugate base, often undefined, which hasa high affinity for metal cations. In example, upon complexation tolanthanide and actinide ions, DTPA exists as the pentaanionic form, i.e.all five carboxylic acid groups are deprotonated. DTPA has a molecularformula of C₁₄H₂₃N₃O₁₀ with molar mass 393.358 g/mole and a chemicalformula as:

Currently, DTPA is approved by the U.S. Food and Drug Administration(FDA) for chelation of three radioactive materials: plutonium,americium, and curium. DTPA is the parent acid of an octadentate ligand,diethylene triamine pentaacetate. In some situations, all five acetatearms are not attached to the metal ion. In one aspect of the presentinvention, DTPA has been conjugated to γ-PGA through hexanediamine((γ-PGA)-DTPA) as illustrated below:

In one aspect of the invention, (γ-PGA)-DTPA is one species of thePGA-complexone conjugates used in the current pharmaceutical compositionof nanoparticles. The overall degree of substitution of DTPA in(γ-PGA)-DTPA conjugate is generally in the range of about 1-70%,preferably in the range of about 5-40%, and most preferably in the rangeof about 10-30%. DTPA does not build up in the body or cause long-termhealth effects.

Nanoparticles comprising chitosan, PGA-complexone conjugates and atleast one bioactive agent using the simple and mild ionic-gelationprocess described herein has demonstrated the desired paracellulartransport efficacy with TEER measurements in the Caco-2 cell culturesmodel as described in Example No. 4.

Example No. 27 Enzyme Inhibition Study with (γ-PGA)-DTPA Conjugate

Brush border membrane bounded enzymes were used to simulate a contactingmembrane at the bottom of a donor compartment, wherein theinsulin-loaded medium (Krebs-Ringer buffer) in the donor compartment wasused as the starting material at time zero. Three elements were used inthis enzyme inhibition study to assess the enzymatic degradation ofinsulin versus time by brush border membrane bounded enzymes. They were(a) insulin 1 mg/ml as control; (b) DTPA 5 mg/ml; and (c) (γ-PGA)-DTPA 5mg/ml. As shown in FIG. 22, both DTPA and (γ-PGA)-DTPA substantiallyprotect or maintain the insulin activity or viable content over theexperimental duration up to 2 hours. Some aspects of the presentinvention provide a pharmaceutical composition of nanoparticles, thenanoparticles comprising a shell portion that is dominated by positivelycharged chitosan, a core portion that comprises complexone and onenegatively charged substrate, wherein the substrate is PGA, wherein thenegatively charged substrate is at least partially neutralized with aportion of the positively charged chitosan in the core portion, and atleast one bioactive agent loaded within the nanoparticles. In oneembodiment, the PGA is conjugated with the complexone to formPGA-complexion conjugates within the nanoparticles.

Some aspects of the invention provide a method of enhancing enzymaticresistance of a bioactive agent in oral administration by encapsulatingthe bioactive agent in nanoparticles, wherein the nanoparticles have apharmaceutical formulation and/or composition as described in thisdisclosure and in claims. In one embodiment, the nanoparticles arefurther loaded with pharmaceutically acceptable carrier, diluent, orexcipient in tablets, pills, capsules, and the like.

Hygroscopy

Hygroscopy is the ability of a substance to attract and hold watermolecules from the surrounding environment through either absorption oradsorption with the adsorbing or absorbing material becoming physically‘changed,’ somewhat: by an increase in volume, stickiness, or otherphysical characteristic of the material as water molecules become‘suspended’ between the material's molecules in the process. While somesimilar forces are at work here, it is different from capillaryattraction, a process where glass or other ‘solid’ substances attractwater, but are not changed in the process, e.g. water molecules becomingsuspended between the glass molecules. Because of their affinity foratmospheric moisture, hygroscopic materials might necessarily be storedin sealed containers or sealed from atmospheric moisture. Chitosan andchitosan-shelled nanoparticles exhibit hygroscopic properties.

Some aspects of the invention relate to means or methods to maintain thechitosan-shelled nanoparticles of the present invention from undesiredatmospheric moisture. In one embodiment, the nanoparticles are coatedwith a coating material, such as the enteric coating polymer thatprevents moisture from seeping or leaking into the sealed nanoparticles.In another embodiment, the nanoparticles are loaded in a capsule that isfilled with a moisture-free gas, such as moisture-free nitrogen CO₂, ornitrogen-based inert gas. In still another embodiment, the nanoparticlesare formed in a pill or tablet configuration that is coated with amoisture-proof coating material or polymer.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

1. A pharmaceutical composition of nanoparticles, said nanoparticlesconsisting of a shell portion that is dominated by positively chargedchitosan, a core portion that consists of said positively chargedchitosan, one negatively charged substrate, at least one bioactive agentloaded within said nanoparticles, and optionally a zero-charge compound,wherein said at least one bioactive agent is an anti-hemophilic factor.2. The pharmaceutical composition of claim 1, wherein said chitosan isN-trimethyl chitosan, EDTA-chitosan, low molecular weight chitosan,mono-N-carboxymethyl chitosan, chitosan derivatives, or combinationsthereof.
 3. The pharmaceutical composition of claim 1, wherein saidnanoparticles are formed via a simple and mild ionic-gelation process.4. The pharmaceutical composition of claim 1, wherein said nanoparticlesare in a tablet or pill configuration.
 5. The pharmaceutical compositionof claim 4, wherein said tablet or pill is treated with an entericcoating.
 6. The pharmaceutical composition of claim 1, wherein saidnanoparticles are freeze-dried, thereby said nanoparticles being in apowder form.
 7. The pharmaceutical composition of claim 1, wherein saidnanoparticles are loaded in a capsule.
 8. The pharmaceutical compositionof claim 7, wherein said capsule further comprises a pharmaceuticallyacceptable carrier, diluent, or excipient.
 9. The pharmaceuticalcomposition of claim 7, wherein said capsule further comprises at leasta solubilizer, bubbling agent, dispersion agent, or emulsifier.
 10. Thepharmaceutical composition of claim 7, wherein said capsule is treatedwith an enteric coating.
 11. The pharmaceutical composition of claim 7,wherein said capsule further comprises at least one absorption enhancer.12. The pharmaceutical composition of claim 1, wherein saidnanoparticles are treated with an enteric coating.
 13. Thepharmaceutical composition of claim 12, wherein said absorption enhanceris selected from the group consisting of bile salts, surfactants,medium-chain fatty acids, phosphate esters, chitosan, and chitosanderivatives.
 14. The pharmaceutical composition of claim 1, wherein saidnegatively charged substrate is PGA-complexone conjugate, γ-PGA, α-PGA,derivatives of PGA, salts of PGA, or combinations thereof.
 15. Thepharmaceutical composition of claim 1, wherein said zero-charge compoundis an absorption enhancer.
 16. The pharmaceutical composition of claim1, wherein said anti-hemophilic factor is a recombinant humancoagulation factor VIIa (rFVIIa) or a recombinant plasma/serum-freecoagulation factor VIII.
 17. The pharmaceutical composition of claim 1,wherein said anti-hemophilic factor is selected from the groupconsisting of Bioclate (manufactured by Aventis Behring), Helixate(manufactured by CSL Behring), Kogenate (manufactured by BayerHealthcare), Recombinate (manufactured by Baxter Healthcare) Advate(manufactured by Baxter Healthcare), Alphanate (manufactured by GrifolsSA), Hemofil-M (manufactured by Baxter Healthcare), Humate-P(manufactured by CLS Behrng), Koate (manufactured by TalecrisBiotherapeutics), Monarc-M (manufactured by Baxter Healthcare),Monoclate-P (manufactured by CSL Behring), and Refacto (manufactured byWyeth).
 18. The pharmaceutical composition of claim 1, wherein saidanti-hemophilic factor is a coagulation factor to treat an animalsubject that lacks functional clotting factor VIII, factor IX, or factorXI.
 19. The pharmaceutical composition of claim 1, wherein saidanti-hemophilic factor is covalently attached with polyethylene glycolpolymer chains.
 20. The pharmaceutical composition of claim 1, whereinsaid anti-hemophilic factor is a glycoprotein procofactor.